Methods and compositions for isolating, quantifying, characterizing, and modulating antigen-specific T cells

ABSTRACT

The present invention concerns artificial antigen presenting cells (aAPCs) and methods of making and using the same, for example, to isolate, identify, and expand T cell populations specifically reactive against a disease-associated antigenic peptide, as well as to modulate responses of antigen-specific T cells both in vivo, ex vivo, and in vitro. Accordingly, the aAPCs of the invention can be used to treat conditions that would benefit from modulation of a T cell response, for example, autoimmune disorders, allergies, cancers, viral infections, and graft rejection. In certain preferred embodiments, the aAPCs are liposomes comprised of MHC:peptide complexes and accessory molecules. Other molecules, such as co-stimulatory molecules and adhesion molecules, can also be included in the compositions of the invention. In other embodiments, the aAPCs are comprised of a scaffold to which a plurality of MHC:peptide complexes and accessory molecules (as well as other molecules) can be attached at high density.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/756,983, filed 19 Jan. 2001, which is a continuation-in-partof U.S. patent application Ser. No. 09/421,506, filed 19 Oct. 1999, andPCT application No. PCT/US99/24666, filed 19 Oct. 1999, to which thisapplication claims the benefit of and priority to. Additionally, thisapplication claims the benefit of and priority to provisionalapplication Nos. 60/105,018, filed 20 Oct. 1998, and 60/510,645, filed10 Oct. 2003. Each of the foregoing patent applications, and any patentissuing from any of them, is hereby incorporated by reference in itsentirety.

GOVERNMENT INTERESTS

This invention was made with government support under NIH Grant Nos.AR40770, A137232, and AR41897. The government may have certain rights inthis invention.

FIELD OF THE INVENTION

This invention concerns immunology. Specifically, the invention relatesto methods of preparing artificial antigen presenting cells, as well ascompositions comprising such artificial antigen presenting cells.Further, the invention concerns the application of such artificialantigen presenting cells to isolate antigen-specific T cells, modulatingT cell responses, and treating conditions which would benefit from themodulation of a T cell response, for example, autoimmune disorders,allergies, cancers, and viral, protozoal, and bacterial infections, aswell as in transplantation therapy.

BACKGROUND OF THE INVENTION

1. Introduction.

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that anysuch information is prior art, or relevant, to the presently claimedinventions, or that any publication or patent specifically or implicitlyreferenced is prior art.

2. Background.

The immunologic arts have advanced markedly in recent years. Thecomplexity of the science explaining aspects of the field is immense.Set forth below is a discussion concerning known aspects of variouselements involved in immunogenic responses and concepts in the art thatare related to the invention disclosed herein.

A. T Cells

T lymphocytes (i.e., T cells) are part of the immune system, whichdefends the body against bacterial, viral, and protozoal infection, aswell as molecules that contain epitopes recognized as non-self(including aberrant forms of molecules that occur naturally in thebody). The recognition of non-self molecules as well as the destructionof infectious agents carrying non-self antigens is a function of Tcells, which, along with certain B lymphocytes (i.e., B cells), providefor the cell-mediated immune responses of adaptive immunity.

Infecting pathogens are generally accessible to extracellular antibodiesfound in the blood and the extracellular spaces. However, some infectingagents, including all viruses, replicate inside cells where they are notexposed to, and thus cannot be detected by, extracellular antibodies. Inorder for these foreign agents to be accessible to the cell-mediatedimmune response, cells harboring such pathogens must either “express”antigenic motifs of the infecting agents on the surface of the cells orthe antigenic motifs must be shed from the cells, e.g., by cell death,to be accessible to and subsequently expressed on the cell membrane ofphagocytic antigen presenting cells (“APCs”) that participate incell-mediated immune processes.

Antigens derived from replicating viruses, for example, are displayed onthe surface of infected cells where they may be recognized by“cytotoxic” T cells or other surveillance cells such APCs, e.g.,dendritic cells. T cells may then respond to the infection byrecognizing the viral antigens and then killing the infected cell. Theactions of such cytotoxic T cells depend upon direct interaction betweenthe antigenic motif of the infecting agent expressed on the surface ofthe infected cells and T cell receptors specific for the motif.

Although T cells are important in responding to intracellularinfections, some foreign agents evade such responses because theyreplicate in the vesicles of macrophages, as occurs with Mycobacteriumtuberculosis, the pathogen that causes tuberculosis. Whereas bacteriathat enter macrophages are usually destroyed in lysosomes (which containa variety of enzymes and bactericidal substances), infectious agentssuch as M. tuberculosis may survive because the vesicles they occupy maynot fuse with macrophage lysosomes. The immune system fights such agentsusing a second type of T cell, known as a “T helper cell,” which helpsto activate macrophages and induce the fusion of lysosomes with vesiclescontaining the infectious agents. The T helper cells also bring aboutthe stimulation of other immune mechanisms of the phagocyte. T helpercells may further be involved in initiating and/or sustaining the immunesystem's release of soluble factors that attract macrophages and otherprofessional APCs to the site of infection.

Additionally, specialized T helper cells play an important role in thedestruction of extracellular pathogens by interacting with B cells.Depending on the type of infection being controlled, participating Thelper cells may have an inflammatory, or Th1-like phenotype, or asuppressive, Th2-like phenotype. Other T helper cell types may alsoexist, including Treg and Th3 cells. In the case of cytotoxic T cellsand Th1 cells, fragments of non-self proteins (i.e., processed antigens)are recognized on the surface of the target cell (such as an infectedcell). Th2 cells, on the other hand, recognize and interact with antigenpresented by professional antigen presenting cells such as dendriticcells and B cells. Dendritic cells non-specifically internalize foreignantigens while B cells may bind and internalize foreign antigens viatheir antigen-specific cell surface immunoglobulin. In any case, T cellsrecognize their targets by detecting non-self antigenic motifs (e.g.,peptide fragments derived from, for example, a bacterium or virus) thatare expressed either on infected cells or other immune cells, e.g.,phagocytic APCs.

The actions of T cells depend on their ability to recognize antigenicmotifs on cells (such as cells harboring pathogens or that haveinternalized pathogen-derived products). T cells recognize peptidefragments (e.g., pathogen-derived proteins) in the form of complexesbetween such peptides and MHC (major histocompatibility complex)molecules that are expressed on the surface of antigen presenting cells.

B. T Cell Receptors

T cell receptors (TCRs) are closely related to antibody molecules instructure, and they are involved in antigen binding although, unlikeantibodies, they do not recogize free antigen; instead, they bindantigen fragments which are bound and presented by antigen-presentingmolecules. An important group of antigen-presenting molecules are theMHC class I and class II molecules that present antigenic peptides andprotein fragments to T cells. Other antigen presenting molecules havealso been identified, including CD 1, which present lipid and glycolipidantigens to T cells.

Variability in the antigen binding site of a TCR is created in a fashionsimilar to the antigen binding site of antibodies, and also providesspecificity for a vast number of different antigens. Diversity occurs inthe complementarity determining regions (CDRs) in the N-terminal domainsof the disulfide-linked alpha (α) and beta (β), or gamma (γ) and delta(δ), polypeptides of the TCR. The CDR loops are clustered together toform an MHC-antigen-binding site analogous to the antigen-binding siteof antibodies, although in TCRs, the various chains each contain twoadditional hypervariable loops as compared to antibodies. TCR diversityfor specific antigens is also directly related to the MHC molecule onthe APC's surface to which the antigen is bound and presented to theTCR.

C. MHC Molecules

In general, T cell responses to “non-self” peptide motifs depend on theinteractions of the T cells with other cells that express of otherwisecontain the proteins recognized as non-self. The molecules thatassociate with these peptide or antigen fragments and present them to Tcells are membrane glycoproteins encoded by a cluster of genes thatcomprise the MHC. In general, the term MHC is used to describe generallythe molecules in the mammalian immune system involved in thepresentation of antigenic motifs to T cells. Here, MHC means any majorhistocompatibility complex class I or class II protein from anymammalian organism, including a human, mouse, rat, hosre, pig, dog, cat,or sheep, that present antigens to T cells. Such molecules includefull-length MHC molecules or subunits thereof, and further includeMHC-encoded antigen-presenting glycoproteins having the capacity to binda antigenic peptides. MHC proteins may comprise an amino acid sequencethat is expressed and purified from natural sources, or by any synthetictechniques such as recombinant expression of a desired gene, or group ofgenes, in prokaryotic or eukaryotic systems, which can, for example,result in different patterns of glycosylation. The MHC sequence maycomprise a natural or modified amino acid sequence, including those thatare truncated at either or both the C— or N-terminus and/or include oneor more amino acid insertions, deletions, or substitutions in anaturally occurring MHC sequence.

The two types of MHC molecules, i.e., MHC class I and MHC class IImolecules, deliver peptides from different sources (class I MHCdelivering intracellularly derived peptides, and class II MHC deliveringextracellularly derived peptides) to the surface of the infected cell.The two classes of MHC molecules vary with respect to the length ofpeptides that they are able to present. The binding pocket of MHC classI molecules is blocked at either end, thereby imposing severerestrictions on the size of peptides it can accommodate (typically about8-10 residues). The binding groove of the MHC class II molecules, on theother hand, allows peptides to protrude from the ends, and consequentlymuch longer peptides (e.g., from about 8-30 residues) can bind.Rudensky, et al. Nature, vol. 353:622-27; Miyazaki, et al., Cell, vol.84:531-41; Zhong, et al., J Exp. Med., vol. 284:2061-66.

A naturally occurring MHC class I molecule comprises a glycosylatedheavy chain non-covalently associated with P2-microglobulin. The class Iheavy chain contains an extracellular portion having three domainstermed α1 (the N-terminal-most domain), α2, and α3, a transmembranedomain of about 25 amino acid residues, and a cytoplasmic tailcomprising 30-40 residues. The extracellular domain is glycosylated,with glycosylation varying depending upon species and haplotype. Each ofthe three a domains is about 90 residues in length. X-raycrystallographic studies of the extracellular domains reveal a platformof eight anti-parallel β strands supporting the helices in α1 and α2domains (one a-helix in each domain) arranged in an anti-parallelfashion. A long groove separates these α-helices, which is believed tobe binding site for processed antigen. The α2 and α3 heavy chain domainseach contain intradomain disulphide bonds. The α3 domain is structurallysimilar to immunoglobulin C domains. β2-microglobulin has the structureof an immunoglobulin constant region domain, and it is essential forcell surface expression of MHC class I molecules.

In contrast, the MHC class II molecules are heterodimers of heavy (α)and light (β) chains encoded by the class genes of MHC (genes A and E inthe mouse, and DP, DQ, and DR in humans), with the peptide bindinggroove being located between the two chains. The heavy chains vary inmolecular weight between 30-34 kDa, whereas the light chains have amolecular weight of about 26-29 kDa, with much of this weight differencebeing due to differences in glycosylation. Despite this, the a and 13chains have the same overall structure, with each comprising anextracellular portion having two domains, α1 and α2 in the a chain andβ1 and β2 in the β chain, followed by short transmembrane region ofabout 30 residues and small cytoplasmic domain of 10-15 amino acidresidues. The α2 and α2 domains are similar to the class I α3 domain andβ32-microglobulin. The β1 domain contains a disulphide bond. The α1, α2,and β1 domains are N-glycosylated, whereas the β2 domain, which containsa CD4 binding site, is not. Class II molecules on APCs interact with CD4molecuels on T cells in an analogous way that that class I moleculesinteract with CD8 molecules. CD4 and CD8 are important in antigenpresentation, which are involved in kinase recruitment that signal Tcell activation.

Although the structures of class I and II molecules are similar, thegroove in class II molecules is more open, and thus can accommodatelonger peptides than the binding groove of class II molecules. For bothclass I and II molecules, the toplogy of the peptide-binding groovedepends in part on the amino acids that comprise the groove, and peptidebinding depends on the nature of the peptide's side chains and theircomplementarity with the binding groove. Garboczi, et al., Nature, vol.384: 134-41; Ward and Quadri, Curr Op Immunol., vol. 9:97-106; Garcia,et al., Science, vol. 279:1166-72).

D. Antigen Processing

Antigens recognized by T cells in the context of an MHC molecule aredegraded or processed so that the determinant recognized by the TCR isonly a small fragment of the original antigen. Antigens are processedinto peptide fragments before association with MHC molecules. TCRs aresensitive to peptides in the MHC groove, rather than the conformationaldeterminant recognized by antibodies.

Peptides bound to MHC class I molecules are recognized by CD8+T cells(cytotoxic T cells), and those bound to MHC class II molecules arerecognized by CD4+T cells (helper T cells). Two functional subsets of Tcells can thereby be activated to initiate the destruction of cells thatpresent, or express, the particular antigen on its surface, and therebyeliminate the source of the antigen (e.g., a pathogen or diseased cell).CD4+T cells may also help to activate B cells, and in turn give rise tothe stimulation of antibody production against the antigenic motifs ofextracellular pathogens.

Infectious agents or other “non-self” antigens can reside in variousintracellular compartments. Viruses and certain bacteria replicate inthe cytosol or in the contiguous nuclear compartment, while manypathogenic bacteria and some eukaryotic parasites replicate in theendosomes and lysosomes that form part of the vesicular system. Theimmune system has different strategies for eliminating such agents fromthese two sites. Cells containing viruses or bacteria located in thecytosol are eliminated by cytotoxic T cells that express thecell-surface molecule CD8 and present antigens on MHC class I molecules.The function of CD8+T cells is to kill infected cells.

Immunogenic agents located in the vesicular compartments of cells aredetected by a different subset of T cells, distinguished by cell surfaceexpression of the molecule CD4. CD4+T cells are specialized toactivate/modulate other cells and fall into at least two primaryfunctional classes: Th1 cells, which activate various immune competentcells to destroy the intravesicular non-self antigenic agents theyharbor; and Th2 cells, which help to activate B cells to, among otherthings, make antibodies against such antigenic agents.

To produce an appropriate response to infectious microorganisms, T cellsmust distinguish self from non-self (e.g., foreign) material coming fromthe different processing pathways. This is achieved by deliveringpeptides to the cell surface from these intracellular compartments usingeither MHC class I or II molecules. As noted above, MHC class Imolecules deliver peptides that come from proteins synthesized in thecell, with the antigen being expressed in association with an MHC classI molecules (antigen:MHC complex) that can be recognized by CD8+T cellsspecific for the particular antigen:MHC complex. In contrast, MHC classII molecules deliver “non-self” peptides derived from proteins that havebeen internalized by the cell (and are thus extracellular from theperspective of the antigen-presenting cell) and processed forpresentation at the cell surface in the context of an MHC class IImolecule. CD4+T cells specific for the particular antigen:MHC class IImolecule complex can then recognize the complex when presented on thesurface of an APC.

E. Antigen Presenting Cells

A wide variety of cells can present antigens. In lymphoid organs, thethree primary APC types are dendritic cells, macrophages, and B cells.Dendritic cells are abundant in in t cell areas of lymph nodes andspleen, and are very effective in initially activiating na{dot over(i)}ve T cells. When na{dot over (i)}ve T cells encounter a specificantigen for the first time on the surface of an antigen-presenting cell(APC) in the context of an appropriate MHC molecule (i.e., priming),they are activated to proliferate and differentiate into cells capableof contributing to the removal of the particular antigen and its source(e.g., an infecting pathogen). The APCs are specialized in that theyexpress surface molecules that synergize with a specific antigen in theactivation of n{dot over (a)}ive T cells. APCs become concentrated inthe peripheral lymphoid organs, to which they migrate after trappingantigen while circulating in the periphery. APCs present peptidefragments to recirculating T cells, some of which may be na{dot over(i)}ve. Dendritic cells are known to present processed antigens tomacrophages, which are involved in the phagocytosis of cells in a firstline of defense against infectious agents. Dendritic cells are also ableto present internalized, processed antigens on both class I and class IIMHC molecules. APCs are also known to be activated by armed effector Tcells. B cells also serve as APCs under some circumstances.

One of the features of APCs is the expression of co-stimulatorymolecules, including the potent B7 molecules, including B7-1 (CD80) andB7-2 (CD86). B7 are constitutively expressed on the surface of dendriticcells, and can be upregulated on monocytes, B cells, and other APCs.They are ligands for CD28, and its homologue CTLA-4 (CD152), which isexpressed after T cell activation. CD28 the primary co-stimulatoryligand expressed on na{dot over (i)}ve T cells. Anderson, et al., J.Immunol., vol. 159:4:1669-75. CD28 stimulation has been shown to prolongand augment cytokine production, and may be important in preventinginduction of tolerance.

The activation of T cells by APCs leads to proliferation of theactivated T cells and to the differentiation of their progeny into armedeffector T cells. The proliferation and differentiation of T cellsdepends on the production of cytokines (such as the T cell growthfactor, IL-2) and their binding to high-affinity receptors on theactivated T cell. T cells whose TCRs are bound to antigens in theabsence of co-stimulatory molecules may fail to make cytokines andinstead may become anergic. This dual requirement for bothreceptor/antigenic interaction and co-stimulation helps to furthermediate na{dot over (i)}ve T cell response.

Proliferating T cells develop into armed effector T cells. Once anexpanded clone of T cells achieves effector function, the T cell cloneprogeny can act on any target cell that displays or expresses a specificantigen on its surface. Effector T cells can mediate a variety offunctions. The killing of infected cells by CD8+ cytotoxic T cells andthe activation of professional APC by Th1 cells together make upcell-mediated immunity. The activation of B cells by both Th2 and Th1cells helps to produce different types of antibodies, thus driving thehumoral immune response. Kirberg, et al., J. Exp. Med., vol.186:8:1269-75.

F. T Cell Activation

T cells generally become sensitized to antigens by becoming trapped inlymphoid organs as the T cells drain into lymph nodes through which theycirculate. Antigens introduced directly into the bloodstream, or thatreach the bloodstream from an infected lymph node, are picked up by APCsin the spleen, for example, where lymphoid cell sensitization occurs inthe splenic white pulp. The trapping of antigen by APCs that migrate tothese lymphoid tissues combined with the continuous recirculation of Tcells through the tissues ensures that rare antigen-specific T cellswill encounter their specific antigen being presented by an APC.Overall, T cell activation has four stages: adhesion, antigen-specificactiviation, co-stimulation, and cytokine signaling.

The recirculation of na{dot over (i)}ve T cells through the lymphoidorgans is orchestrated by non-specific, rapidly reversible adhesiveinteractions between lymphocytes and endothelial cells. Na{dot over(i)}ve T cells enter the lymphoid organs through a process that isthought to occur in a number of steps. The first step in this process ismediated by selectins expressed on the T cell. For example, L-selectinon na{dot over (i)}ve T cells binds to sulfated carbohydrates on thevascular addressins GlyCAM-1 and CD34. CD34 is expressed on endothelialcells in many tissues but is properly glycosylated for L-selectinbinding only on the high endothelial venule cells of lymph nodes.L-selectin binding promotes a rolling interaction, which is critical tothe selectivity of na{dot over (i)}ve lymphocyte homing. Although thisinteraction is too weak to promote extravasation, it is essential forthe initiation of the stronger interactions that then follow between theT cell and the high endothelium, which are mediated by molecules with arelatively broad tissue distribution. (Finger, et al., Nature, Vol.379:266-9).

Stimulation by locally bound chemokines activates the adhesion moleculeLFA-1 on the T cell, increasing its affinity for ICAM-2, which isexpressed constitutively on all endothelial cells, and ICAM-1, which, inthe absence of inflammation, is expressed only on the high endothelialvenule cells of peripheral lymphoid tissues. The binding of LFA-1 to itsligands, ICAM-1, ICAM-2, and ICAM-3, plays a major role in T celladhesion to and migration through the wall of the blood vessel into thelymph nodes. Bachmann, et al., Immunity, vol. 7:549-57.

The high endothelial venules are located in the lymph nodes. This areais inhabited by dendritic cells, which have recently migrated from theperiphery. The migrating T cells scan the surface of these APCs forspecific antigen:MHC complexes. If they do not recognize antigenpresented by these cells, they eventually leave the node via an efferentlymphatic vessel, which returns them to the blood so that they canrecirculate through other lymph nodes. Rarely, a n{dot over (a)}ive Tcell recognizes its specific antigen:MHC complex on the surface of anAPC, which then signals the activation of LFA-1, causing the T cell toadhere strongly to the APC. Binding to the antigen:MHC complex alsoactivates the cell to proliferate and differentiate, resulting in theproduction of armed, antigen-specific T cells. The number of T cellsthat interact with each APC in lymph nodes is very high, as can be seenby the rapid trapping of antigen-specific T cells in a single lymph nodecontaining antigen.

G. Identification and Isolation of Antigen-Specific T Cells

In addition to their role in combating infections, T cells have alsobeen implicated in the destruction of cancerous cells. Autoimmunedisorders have also been linked to antigen-specific T cell attackagainst various parts of the body. One of the major problems hamperingthe understanding of and intervention on the mechanisms involved inthese disorders is the difficulty in identifying T cells specific forthe antigen to be studied. Accordingly, it is of great interest to beable to identify antigen-specific T cells. Additionally, it would be ofgreat therapeutic benefit if T cells specific for a particular antigencould be (i) enriched and then reintroduced in a disease situation, (ii)selectively depleted in the case of an autoimmune disorder, or (iii)modified to alter their functional and/or phenotypic characteristics.Thus, identification and isolation of antigen-specific T cells is anessential requirement in immunology and medicine to understand andmodulate immune responses.

Identification of antigen-specific T cell populations is generallyaccomplished by indirect means in animal models, such as by evaluatingmembrane markers correlated to activation or maturation of these cells.This has usually been accomplished using transgenic systems (Ignatowicz,et al., Cell, vol. 84:521-29; Sebzda, et al., Science, vol. 263:1615-18;Jameson, et al., Ann. Rev. Immunol., vol. 13:93-126). Analysis isgenerally done by means of flow cytometry, where a detector on a machineis capable of identifying cells bound to fluorescent substrates, such asfluoresceinated antibodies. Positively identified cells can be sortedfor further use. Quantitation and isolation of antigen-specific T cellsis usually accomplished by limiting dilution and cloning techniques.When using sorted cells, these approaches become quite cumbersome andare sometimes inaccurate, since the biological effects of antigenrecognition can spread beyond the cells recognizing the antigen. Forinstance, upon engagement of the specific MHC:antigenic peptide complex,T cells produce cytokines that can affect expression of the same markersof activation in non-specific bystander T cells. Hence, in order toisolate and characterize cells with specificity for a given antigen,alternative procedures, such as T cell cloning, need to be applied.These techniques often require many months of technical proceduresbefore results can be obtained. The rate of success, in particular forhuman systems, is quite low, and the population selected may notnecessarily represent the biologically relevant component of the immuneresponse to a given peptide. The direct interaction of a specific T cellwith the antigen:MHC complex would thus be a preferred basis for T cellisolation.

H. Distinctions

Kendrick, et al., U.S. Pat. No.5,595,881, discuss a method for thedetection and isolation of MHC:antigen-restricted T cells. That methodis performed by preparing a MHC:antigen complex isolated using metalchelating technology. The complex is then bound to a planar solidsupport (i.e., a glass coverslip), followed in turn by combining theimmobilized complex with a biological sample so that the MHC:antigencomplex may bind to and retain antigen-specific T cells. Determinationof the presence of reactive MHC:antigen complexes is carried out bydetecting cell proliferation.

The method of Kendrick, et al. substantially differs from the currentinvention. First, the MHC components of the complexes discussed in theKendrick, et al. disclosure are immobilized on a planar solid support.In contrast, the MHC components of the current invention are not boundto a solid planar support. Indeed, in certain preferred embodiments ofthe instant invention, the MHC components are “free floating,” i.e.,they are capable of laterally diffusing, within the fluid lipid bilayerof a liposome membrane, preferably one comprised of phosphotidylcholineand cholesterol components, as described in greater detail below. Thisdifference is substantial in that the MHC:antigen complexes discussed inthe Kendrick, et al. disclosure are believed not to be able toparticipate in the migration or concentration of such complexes in“capping,” which is important to improved binding and activation ofbound T cells. Second, the method discussed in the Kendrick, et al.disclosure concerns detecting “natural” APCs specific for pre-selectedantigen-specific T cells. Reportedly, this is accomplished by isolatingantigen-specific T cells by first performing a series of steps includingbinding antigen via a metal chelating process to a solid support, usingthe antigen to capture antigen-specific MHC components, and thenisolating the MHC:antigen complexes which are in turn bound to a planarsolid support via a linker.

The current invention is much more versatile. It is not concerned withdetecting natural APCs; instead, it concerns the isolation andmanipulation of antigen-specific T cells. The manipulation of such Tcells can be carried out for numerous reasons, such as to directlyimpact T cell function by modulating a T cell response. Suchmanipulations can be performed in vivo, in a column format (whereartifical APCs are bound to a solid support, for example), or insolution, for example, via flow cytometry techniques such as FACS(fluorescence-activated cell sorting). The compositions of the currentinvention can modulate T cell function by including various functionalmolecules into the APC. For example, in one embodiment of the currentinvention, known MHC molecules may be incorporated into liposomes alongwith a labeled antigenic peptide for which such MHC has specificity(e.g., in the case of FACS, a biotinylated antigen). Theliposome:MHC:biotinylated antigen complex may be used to bind toantigen-specific T cells. Binding can be visualized by FACS, followed bysorting the bound cells. Thus, no cell proliferation assay is necessaryto identify and isolate antigen-specific T cells.

In addition to the MHC:antigen complex, the artificial APCs of thisinvention that are used to capture antigen-specific T cells preferablyinclude accessory molecules to help stabilize the MHC:antigen:TCRinteraction, and may also include functional molecules such asco-stimulatory molecules which, in some embodiments, may be used toactivate T cells; adhesion molecules, which may be used to bind cellsdestined for a certain area of the body; targeting molecules that targetthe aAPC to a specific cell or tissue type in a patient's body; andother accessory or functional molecules such as cytokines or antibodiesto cytokine receptors, which are known to have immunomodulatory effectsupon T cells. Moreover, in some embodiments the current inventionfurther provides for properly orienting these molecules in aliposome-based aAPC through the use of a novel anchoring mechanism, apreferred example of which comprises a GM-1 ganglioside molecule and theβ subunit of cholera toxin. In this context, the protein of interest maybe connected to a cholera toxin subunit as a fusion protein or by use ofa linking moiety. By attaching the cholera toxin subunit to the moleculeof interest, the cholera toxin may be bound by the GM-1 gangliosidemolecule incorporated into lipid membrane of the liposome, as the GM-1ganglioside molecule has affinity for the nonpolar region of themembrane.

In liposome-based embodiments of the invention, all of these moleculesmay be incorporated into the lipid bilayer of the liposome. Given thefluidity of the membrane, the molecules embedded therein can laterallydiffuse and become concentrated over time, for example, at an aAPC-Tcell interface, to form a structure analogous to the immunologicalsynapse that forms between T cells and APCs. Further, other moleculesmay be included in the compositions that do not influence the modulationof T cell responses. Examples of such molecules include proteins usefulin anchoring the artificial APC to a solid support, molecules fororienting other molecules to be included in an aAPC, as well as proteinsor other molecules that target an aAPC to a cell or tissue within apatient's body. As used herein, such molecules that are not associatedwith modulation or T cell binding are termed “irrelevant” molecules.

Additionally, a label may be attached (covalently or non-covalently) tothe antigen, an irrelevant molecule, or another aAPC component, forexample, to a lipid within a liposome. The artificial APCs of theinvention also allow for optional expansion of T cell populationsspecific for the MHC:antigen complexes using solution-based (e.g.,roller bottle or bioreactor) cell culture.

The current invention represents a substantial and heretoforeunrecognized advance in the MHC:antigen complex—T cell-binding art inthat the artificial APC is not restricted to complexes of MHC:antigenalone or bound to a planar surface. The importance of the structuraldifferences can not be over emphasized. The addition of accessorymolecules, as well as co-stimulatory molecules, and other proteins inproper orientation (particularly in embodiments that employ liposomes)allow for substantially improved binding association and manipulation ofT cells, which is very important in the identification and stimulationof antigen-specific T cells. This is especially true in solution-basedFACS analysis where the functionality of antigen-specific T cells can beinterpreted directly. For example, prior studies (Watts, T. H., Annalsof the New York Academy of Sciences vol. 81:7564-7568.) respecting themodulation of T cells may be erroneous. There, it was demonstrated thatplanar membranes containing purified MHC loaded with antigen fused toglass cover slips elicited IL-2 production by T cells through theinteraction of the T cell with the MHC:antigen complex. It was alsoshown that the same complex when formed in unilamellar vesicles (i.e.,liposomes) elicited no response. Contrary to such teaching, the instantinvention is based in part on the discovery that liposome vesiclescontaining MHC:antigen complexes can in fact elicit strong responseswhen combined with accessory molecules such as LFA-1, and othermolecules such as co-stimulatory and adhesion molecules. We based ourtheory that liposomes could function without use of a planar array onthe observation (by the same study cited immediately above) that crudemembrane preparations of cellular material from which the MHC waspurified were effective in eliciting T cell responses in both planar andvesicular forms. Subsequently, we discovered that “extraneous” matterexisting in cell extracts that might be hypothesized to impartfunctionality to vesicular forms of lipid bilayers (as opposed tounilamellar liposomes alone) are not important to T cell binding andresponse. Rather, T cell binding and response is possible usingvesicular forms of liposomes containing specific molecules applied incombination with lipsomes (e.g., accessory molecules, co-stimulatorymolecules, and adhesion molecules).

Prior research has also been inconclusive respecting the use of MHCmolecules. For example, it has been shown (Buus, S., Cell, vol.47:1071-1077) that a particular antigenic peptide binds solely to thealpha chain of the class II MHC IAd molecule, while other investigationshave shown that binding interactions between T cell receptors andMHC:antigen ternary complexes use whole MHC, not just single chains ofthe MHC, to determine peptide sequence motifs. Exactly how much of aMHC:antigen complex must be present is not absolutely known and may varywith T cell specificity. The instant invention preferably uses eitherwhole MHC molecules or those parts of the a and 0 subunits of class Iand class II MHC needed to form peptide-binding cleft regions.

The current invention's use of co-stimulatory, adhesion, and otheraccessory molecules in a “free floating” format (in its liposomeembodiments) also helps to both anchor and direct the interactionbetween MHC:antigen:accessory molecule and T cell receptors by providinga structure that mimics those found in the natural state. Specifically,in the liposomes used in the invention, the MHC:antigen:accessorymolecule complexes in conjunction with other functional molecules areable to migrate in proper orientation in the lipid bilayer of theliposome. Such fluidity allows various components within the membrane,which may be randomly distributed prior to interaction with a T cellspecific for the particular antigen being presented, to rapidly becomeconcentrated at the T cell-liposome interface in a manner analogous tothat which occurs between a T cell and an APC in situ during theformation of an immunological synapse. In preferred embodiments, theliposomes of the invention comprise preferred combinations of lipids andsurfactant molecules, particularly phosphotidylcholine and cholesterol.Such aAPC embodiments provide protein presentation characteristics andprotein migration properties similar to those of naturally occurringAPCs, and allow the MHC:antigen complexes to easily migrate to T cellbinding loci similar to “capping” events seen in natural APCs. Moreover,as representively illustrated in the figures, the structure of theliposome-based aAPCs on the invention allows for specific “capping” ofthe liposomes on the surface of the T cells to which the liposomes arebound. Additionally, interaction between the T cell and aAPC-associatedmolecules is further enhanced by the molecules being oriented in thelipid membrane such that their active sites are positioned facingoutward on the APC. Without such orientation, the ratio of properlyoriented molecules to improperly oriented molecules would be expected tobe around 50:50. This ratio is greatly increased using MHC, functional,and accessory proteins that have attached thereto (either by fusionprotein construction or by use of a linker) an orienting moiety, apreferred example of which is cholera toxin β subunit placed in relationto the active center of the protein of interest such that upon the βsubunit being bound by a GM-1 ganglioside molecule incorporated in thelipid layer of the aAPC, the protein of interest will be oriented on theouter surface of the APC, as opposed to being placed in the lumen (i.e.,the internal volume defined by the liposome) of the aAPC.

Additional versatility is available with the current invention in thatthe artificial APCs may incorporate irrelevant molecules to be used, forexample, in conjunction with separate solid support-based capturemoieties for capturing generic target motifs such as irrelevantmolecules. Because of the capacity for the functional molecules tomigrate in the liposome, in such embodiments the irrelevant moleculescan be used to anchor the aAPCs to a solid substrate (e.g., a columnmatrix useful for column chromatography) in a manner that will notinterfere with binding of T cells having TCRs specific for the MHC:antigen complex carried by the aAPCs. As will be appreciated, such asystem avoids the need for manufacturing specialized solid phase capturesubstrates for each antigen-specific complex.

With regard to the capture of an aAPC by a solid phase component (e.g.,a column matrix having one member of a binding pair bound thereto), thetarget molecules used in the aAPC for binding to capture molecules(e.g., the other member of the particular binding pair) of the solidsupport are called “irrelevant” molecules because they do not impact theaAPC:T cell interaction. Such a design further preserves the ability ofthe other molecules present in the aAPC to participate in T cell cappingor activation. In liposome-based embodiments, the complexes involved insuch activities can diffuse away from the irrelevant molecules involvedin column binding. With regard to aAPC embodiments that do not providemembrane fluidity, it is preferred that the irrelevant molecules besegregated to one region of the structure, with the complexes intendedfor T cell interaction being disposed in a different region.

It has been recognized that the number of receptors on a T cell isvariable (Rothenberg, E., Science, vol. 273:78-79). It is also knownthat the number of TCRs and combination of co-stimulatory molecules andaccessory molecules varies with the maturation of the T cell (Dubey, C.,J Immunol., vol. 157:3820-3289). How many such receptors are needed inany situation to elicit a T cell response is unknown. Moreover, it isknown that presence of a co-stimulatory signal decreases the number ofreceptors necessary to activate a T cell (Viola, A. and Lanzavecchia,A., Science, vol. 273:104-106). That said, because the instant inventioninvolves the preparation of aAPCs of defined compositions, it is nowpossible to control the number of MHC:antigen:accessory moleculecomplexes relative to other molecules, including functional moleculessuch as co-stimulatory and adhesion molecules, present in a given aAPCpreparation. Accordingly, the binding and modulation of the T cellresponse at different stages of cell maturation may be “fine tuned”using the instant invention.

In another system, Nag, et al. (U.S. Pat. No. 5,734,023) disclosed MHCsubunits that were complexed with antigenic peptides and “effector”molecules wherein such complexes were used to identify T cellpopulations that were associated with autoimmune diseases. The complexeswere reportedly used to destroy and anergize such T cell populationsfrom a patient's blood cell population. Therein, the effector moleculeswere disclosed as being toxins, radiolabels, etc. which may beconjugated to the MHC or antigen portion of the complexes to effecutatethe identification, removal, anergy, or death of such T cellpopulations. Such effector molecules were not disclosed as being relatedto the attractive binding interactions or T cell responses to effectuatea phenotypic change in the cells. Instead, they were merely designed andintended to aid in the recognition and/or destruction of specific T cellpopulations. Additionally, the Nag, et al. disclosure uses lipids in theconstruction of micelles that are designed for intravenous injection astherapeutics.

The use of negatively charged acidic phospholipids (such asphosphatidylserine) and the lack of cholesterol or GM-1 gangliosidemolecules and cholera toxin subunit in the design of such micellesdiffers from that of the current invention in substantial ways. Forexample, preferred embodiments of the instant invention use neutrallycharged phospholipids such as phosphotidylcholine (Pc). It has beendiscovered that the aAPCs of the instant invention have substantiallyincreased stability because of the Pc and cholesterol in environmentswhere IL-1 is present. IL-1 is known to interact with chargedphospholipids and to destabilize liposome structure. Likewise, inenvironments where TNF is present, the permeability of liposomescomprised of charged phospholipids (e.g., phosphotidylserine) is greatlyaffected. In the same manner, environments where RNase is present mayalso affect charged phospholipid liposome structures. In the presentinvenion, liposome-based embodiments avoid such disruptive effectsthrough the use of neutral phospholipids.

Additionally, the Nag, et al. disclosure provides no insight with regardto the inclusion of co-stimulatory molecules, adhesion molecules, orprotein orientation mechanisms (such as the binding of cholera toxin byGM-1 ganglioside), etc.

In yet another recent disclosure, Spack, et al. (U.S. Pat. No.5,750,356) report a method for monitoring T cell reactivity using amodified ELISPOT assay which detects various factors produced by thestimulation of T cells with numerous factors in the presence of naturalantigen presenting cells. The current invention is distinguishable fromthe Spack, et al. disclosure in that the current invention usesartificial antigen presenting cells that, in addition to peptide loadedMHC complexes, include one more of various accessory, orienting,co-stimulatory, adhesion, cytokine, and/or chemokine molecules that caneffect binding and/or modulation of T cell responses. Additionally, inembodiments that require solid support binding, the aAPCs of theinvention include irrelevant molecules.

In still another disclosure, Wilson, et al. (U.S. Pat. No. 5,776,487)disclose the use of liposome structures for detecting analyte in a testsample, wherein the liposome contains only an analyte-specific ligandand a haptenated component used to bind to a receptor moiety on a solidphase, thereby allowing capture of a test analyte on a solid support fordetection. Thus, it is vastly divergent from the concept of the currentinvention.

The present invention radically advances the art by providing syntheticcompositions that enable, among other things, the stimulation andmodulation of the activity of antigen-specific T cells. This has beenaccomplished by providing structures (e.g., liposomes and dendrimers)that allow high local concentrations of the molecules necessary toeffect modulation of T cell activity to become localized at an aAPC-Tcell interface and thereby faciltate binding and T cell capping. Cappingis the phenomenon by which the T cell focuses the relevant molecules tothe portion of the cell surface where binding has occurred, thusamplifying the binding, and subsequently the signaling of the event tothe cell's other components.

SUMMARY OF THE INVENTION

An object of the invention is to provide compositions useful foridentifying, isolating, modulating, and quantifying antigen-specific Tcells. Other objects provide methods for isolating antigen-specific Tcells using such compositions, as well as using such compositions invivo, in vitro, and ex vivo to modulate the activity of antigen-specificT cells.

In this invention, the aAPCs present antigens complexed with MHCmolecules. The MHC:antigen complexes are presented for contact with, andrecognition by, a T cell receptor specific therefor. Such antigens canbe selected from the group consisting of a peptide, a peptide associatedwith the onset of graft versus host diseases, a cancer cell-derivedpeptide, a peptide derived from an allergen, a donor-derived peptide, apathogen-derived molecule, a peptide derived by epitope mapping, aself-derived molecule, and a self-derived molecule that has sequenceidentity with a pathogen-derived antigen in a range selected from thegroup consisting of between 5 and 100%, 15 and 100%, 35 and 100%, and 50and 100%.

A. Artificial APCs and aAPC Content

An aspect of the invention concerns aAPCs and compositions comprisingthe same. In general, the aAPCs of the invention serve to presentspecific antigenic peptides to TCRs specifically reactive therewith inmanner that can be used either to isolate a T cell specific for theantigen being presented, or, alternatively, to modulate the activity ofsuch T cells and/or provide a mechanism for activating an endogenouscellular immune response for the treatment of disease. This isaccomplished by providing a plurality of antigen-presenting molecules,particularly MHC class I or class II molecules, loaded, or complexed,with an antigenic peptide of interest in conjunction with a plurality ofaccessory molecules in a manner that allows at least some of thesecomplexes to interact with corresponding receptors (e.g., TCRs), bindingpartners, or ligands on the surface of T cells. A number of othermolecules can also be present on the surface of the aAPCs of theinvention, including adhesion molecules and so-called “irrelevant”molecules (e.g., molecules involved in allowing an aAPC to bind to acolumn matrix, molecules that target an aAPC to a specific cell ortissue type in a patient, etc.). Particularly preferred additionalmolecules are co-stimulatory molecules, e.g., B7 molecules.

In certain preferred embodiments, the features of the invention may beprovided by membrane-bound vesicles, particularly liposomes comprised ofa lipid bilayer that exhibits membrane fluidity when placed underphysiological conditions (i.e., those ionic, pH, temperature, and otherconditions that are required in order for T cells to survive). Preferredliposomes are those wherein the fluid lipid bilayer comprises neutralphospholipids, and preferably a surfactant. A particularly preferredneutral phospholipid is phosphotidylcholine, and a particularlypreferred surfactant is cholesterol. In still other embodiments, theaAPCs comprise neutral phospholipids, cholesterol, and GM-1 gangliosidemolecules each present in an appropriate ratio to allow free lateraldiffusion of molecules of interest through the lipid layer, i.e., afluid lipid bilayer.

Such aAPCs further comprise on their exterior surface multiples of homo-or heterogenous combinations of MHC:antigenic-peptide complexesincorporated therein and may comprise other functional moleculesincluding accessory molecules, co-stimulation molecules, adhesionmolecules, and other immunomodulatory molecules such as cytokines,cytokine receptors, chemokines, and chemokine receptors. Such aAPCs mayalso include molecules to provide a desired orientation (e.g., presenceon the exterior, as opposed to the interior, of the aAPC) to, orotherwise anchor, the functional molecules of interest in the aAPC.Vesicles, such as liposomes, can also serve as delivery vehicles for oneor more pharmacological agent species such as nucleic acids (e.g.,expression vectors encoding one or more genes of interest under thecontrol of expression elements, anti-sense oligonucleotides, smallinterfering RNAs, ribozymes, and triplex-forming oligonucleotides thatcan function in a cell that engulfs the liposome), small molecule drugs,therapeutic proteins, proteins which serve to modulate (i.e., enhance ordiminish) T cell stimulation or activation, etc.

In addition to liposomes that comprise a lipid bilayer, some embodimentscomprise a lipid monolayer layered onto a non-planar, preferably aspheroid, solid support such as a plastic (e.g., polystyrene), glass, ormagnetic bead. The beads may be of any suitable size depending uponapplication. Typically, said beads may have a diameter of about 1 um toabout 300 um, although smaller beads may also be used as they provide agreater surface area relative to volume (such beads may be especiallyuseful for in vivo applications where physical constraints,such as theinternal diameter of capillaries, etc., are also important to consider).The solid support should have affinity for non-polar regions of aphospholipid. In other embodiments, the aAPCs of the invention may besynthesized on a scaffold that can be readily modified to include aplurality of different molecules of different types, e.g., MHC class Ior class II molecules complexed with the corresponding antigenic peptideand associated with one or more of the various accessory molecules,co-stimulatory molecules, and irrelevant molecules (e.g., targetingmolecules, tagging or detection molecules, separation molecules, etc.).Examples of such scaffolds include dendrimers and small particles (e.g.,plastic or glass beads, magnetic or metal particles, etc.).

The aAPCs of the invention, in addition to a vesicle or scaffold towhich may be attached multiple MHC molecules complexed with antigenicpeptides, also may include a plurality of accessory molecules of thesame or different species. Said accessory molecules may facilitate andstabilize the interaction between the antigen-specific T cell and theaAPC. A representative example of an accessory molecule is LFA-1. Otheraccessory molecules include, but are not limited to, CD11a/18,CD54(ICAM-1), CD106(VCAM), and CD49d/29(VLA-4), as well as antibodies(or antibody fragments) to ligands for each of these molecules.

With regard to co-stimulatory molecules, which function to helpstimulate, prime, and/or activate (e.g., induce cell proliferation,cytokine production, etc.) an antigen-specific T cell, representativeexamples include, but are not limited to, B7-1, B7-2, CD5, CD9, CD2,CD40, and antibodies to their respective ligands. Such co-stimulatorymolecules can be produced by recombinant methods, as is the case withregard to the other proteins included in the aAPCs of the invention.Co-stimulatory molecules can be used for a variety of purposes inaddition to eliciting cell proliferation. For example, it is known thatmemory CD4+T cells express B7-2, whereas na{dot over (i)}ve CD4+T cellsdo not. (Hakamada-Taguchi, R., European Journal of Immunology, vol.28:865-873). Thus, aAPCs, according to the invention, can be used toselectively target memory T cells by incorporating anti-B7-2 into theartificial APC.

Artificial APCs of the invention can also include adhesion molecules tofacilitate strong and selective binding between the aAPC andantigen-specific T cells. Suitable adhesion molecules include, but arenot limited to, proteins of the ICAM family, for example, ICAM-1 andICAM-2, GlyCAM-1, as well as CD34, anti-LFA-1, anti-CD44, and anti-beta7antibodies, chemokines, and chemokine receptors such as CXCR4 and CCR5,and antibodies to selectins L, E, and P. Such molecules may alsofunction as homing molecules for cells destined for specific locationsin vivo. For example, αβ,7 and L-selectin have been proposed as gut andperipheral lymphnode homing molecules, respectively. The former isexpressed mainly on memory T cells, while L-selectin is expressed mainlyon na{dot over (i)}ve T cells (Abitorabi, M. A., J Immunol., vol.156:3111-3117). Endothelial selectins (E-selectin and P-selectin) areassociated with the extravasasion of T cells into inflammatory sites inthe skin (Tietz, W., J Immunol., vol. 161:963-970). In the currentinvention a P7 binding molecule and gut addressin MAdCAM-1, or ananti-L-selectin antibody, can be incorporated into the aAPCs inassociation with the MHC:antigenic peptide complex.

It is also known that CD44, which binds hyaluronan, is involved inleukocyte extravasation. Anti-CD44 antibody binds to CD44 and strips itfrom the leukocyte surface. In some embodiments, anti-CD44 can beincorporated into an aAPC to strip CD44 from leukocytes as desired,thereby helping to inhibit the extravasation of the cells intoextracellular spaces, for example, once the aAPC-treated cells arereturned to the patient. In another embodiment, anti-CD44 can be infusedinto an immunomodulatory column where the leukocytes have been capturedby artificial APCs for the same purpose.

In some embodiments, other functional molecules (i.e., modulationmolecules) may also be incorporated into aAPCs to facilitate T cellmodulation. Examples of such molecules include, but are not limited to,CD72, CD22, and CD58 (or antibodies to their ligands) antibodies tocytokine or chemokine receptors, or small molecules that mimic theactions of the various cytokines or neuropeptides. Such modulatorymolecules can be used, for example, to modulate antigen-specific Tcells.

In still other embodiments, aAPCs may also comprise irrelevantmolecules, which are included for the purpose of providing a way toanchor the aAPC to a solid support or to carry a label. Such moleculesare termed “irrelevant” because they do not participate in T cellbinding, activation, or modulation.

In still further embodiments, any of the aforementioned molecules ofinterest (e.g., MHC, functional, accessory, irrelevant, etc.) can bebound to an anchor moiety resident in the vesicle membrane. An exampleof such an anchor is cholera toxin β subunit moiety, which can be linkeddirectly or indirectly to a protein of interest by either a linkingmoiety or by a recombinant construction of a fusion protein wherein thetoxin subunit is linked directly thereto during translation. In suchembodiments, the cholera toxin subunit may be positioned with respect tothe molecule of interest such that the active portion of the molecule ofinterest is available for contact with T cells while the toxin portionremains in the the nonpolar region of the lipid layer of the aAPC. Thecholera toxin moiety may remain in the aAPC's interior by binding toGM-1 that is incorporated into the APC's lipid interior.

In still other embodiments the aAPCs comprise a label. Labels can beassociated with any suitable molecule type present in the aAPC,including any of the group consisting of a molecule in the lipid bilayerof a liposome, a lipid of the liposome, an antigen, an MHC molecule, aco-stimulatory molecule, an adhesion molecule, a cell modulationmolecule, a GM-1 ganglioside mlecules, cholera toxin β subunit, anirrelevant molecule, and an accessory molecule. A wide variety of labelsmay be employed, such as radionuclides (e.g., gamma-emittingradioisotopes such as technetium-99 or indium-111), fluorescers (e.g,fluorescein), enzymes, enzyme substrates, enzyme cofactors, enzymeinhibitors, chemiluminescent compounds, bioluminescent compounds, etc.Those of ordinary skill in the art will know of other suitable labelsfor use in practicing the invention.

Closely related aspects of the invention concern compositions anddevices that comprise aAPCs of the invention. For example, certainembodiments of the invention concern pharmaceutical compositionscomprising aAPCs of the invention in a pharmaceutically acceptablecarrier. Such compositions may include both liquid and dry formulations.Similarly, the aAPCs of the invention may be used to make devices toisolate, or capture, antigen-specific T cells. In such devices, aAPCsmay be attached to a solid support. For example, when a solutioncontaining T cells specific for the antigen (as may be represented by adevice that contains one or more different aAPC species specific for theparticular antigen) is passed over the support, the antigen-specific Tcells may be retained. Later, the antigen-specific T cells may berecovered from the solid support. The T cells may then be subjected toadditional manipulations, if desired, including expansion, activation,stimulation, and/or modulation, after which they may be reintroducedinto a patient. Alternatively, such devices can be used to removeparticular antigen-specific T cells from a sample prior to refusion ofthe sample back into a patient.

Kits containing such compositions and devices are also within the scopeof the invention. In general, such kits contain the particularcomposition or device packaged in a suitable manner in an appropriatecontainer and include required package inserts, directions for use, andthe like.

B. Artificial APC Formation

Another aspect of the invention concerns making artifical APCs. Anysuitable process can be used. Genrally, aAPCs are assembled by firstseparately obtaining the various components to be included in the aAPC.Protein and peptide components can, for example, be synthesized byrecombinant techniques, by synthetic chemical methods, or be purchased.Similarly, scaffolds can be synthesized or purchased, as can thecomponents needed to make liposomes. After acquiring the variouscomponents, they can be assembled into the desired aAPC using methodswell known in the art. Of course, the particular composition of aparticular AAPC will depend upon many factors, including the intendedapplication, the availability of alternative reagents, etc.

Liposome-based artificial APCs may be made by obtaining the MHC:antigencomplexes of interest. These complexes are then combined with accessorymolecules (e.g., ICAM-1), lipids and other components, if any (e.g.,cholesterol, anchor molecules, functional molecules, irrelevantmolecules, etc.), to form membrane-associated MHC:antigen:accessorymolecule complexes (i.e., liposome:MHC:antigen:accessory moleculecomplexes). The exact order of addition of the various components willvary depending upon the desired composition, the intended application,etc. It is preferred to combine steps to increase the efficiency andyield of the assembly process. Similar methods may be used to produceaAPCs having a a lipid monolayer, e.g., those having a lipid monolayerlayered on a spheroid solid support.

C. Methods of Use

The invention also concerns methods for using aAPCs. In one aspect, theinvention concerns methods of isolating T cells specific for particularantigens of interest. Such T cells may then be expanded, primed,stimulated, and/or modulated. Furthermore, they may be reinfused into apatient. In another aspect, the invention relates to in vivo methods forstimulating and/or modulating T cell responses. Thus, the inventionconcerns both in vitro, ex vivo, and in vivo methods of treatingconditions that would benefit from the modulation of T cell responses,for example, transplantation therapies, autoimmune disorders, allergies,cancers, infections, and virtually any T cell-mediated disease.

Turning to embodiments of the invention that concern methods ofisolating T cells specific for an antigen of interest, such methods mayinvolve contacting a biological sample containing T cells (some of whichmay or may not be specific for the antigen of interest) with an aAPC,according to the invention, in which the antigen-presenting moleculepresents an antigenic peptide of the antigen of interest. Any resultingaAPC:T cell complexes may then be isolated by separating the T cellsfrom the complexes using any suitable technique. Optionally, thequantity of T cells specific for the particular antigen of interest maybe determined. In addition, or alternatively, the functional phenotypeof the isolated T cells may be determined. Biological samples containingT cells specific for an antigen of interest may include bodily fluidssuch as blood, blood plasma, and cerebrospinal fluid. Other suitablebiological samples include solid tissue, for example, histologicalspecimens.

In such methods, FACS technology may be used. Different components ofthe assay may be labeled e.g., the antigenic peptide may be labelled.Such labels include biotin, fluorochromes, and radioactive labels. Forexample, one type of label that may be used is vancomycin (Rao et al.,Science, vol. 280:5364:708-11, 1998). In other embodiments of suchFACS-based methods, a component of the liposome (or dendrimer orparticle) may be labeled. A label may even be noncovalently enclosedwithin the liposome matrix, for example. If the label is within theliposome, the label may be either enclosed within the liposome orincorporated within the lipids of the outer membrane of the liposome. Inother embodiments, an irrelevant molecule, if present, may be labeled.In yet other embodiments, an aAPC:T cell complex may be removed from abiological sample by capturing the complex via an irrelevant molecule.In such embodiments, a solid support may comprise an irrelevant moleculebinding or capture molecule (e.g., anti-irrelevant molecule antibody)bound to the solid support.

Other methods for isolating T cells specific for an antigen of interestinvolve contacting a biological sample that may or may not contain Tcells with a solid support to which may be attached aAPCs according tothe invention. Resultant aAPC:T cell complexes specific for the antigenof interest may then be isolated, for example, by eluting the T cellsthat bind to the aAPCs on the column. Such affinity chromatographytechniques are well known in the art, and any suitable technique canreadily be adapted for use in conjunction with this invention.

A varierty of kits for isolating T cells specific for an antigen ofinterest are provided. Certain of these embodiments comprise aAPCsaccording to the invention and solid supports, as well as othermaterials that facilitate the completion of the isolation of anantigen-specific T cell population including, but not limited to,solutions such as buffers and culture medium (which solutions mayfurther contain cytokines, antibodies to various transmembrane orsoluble molecules, chemokines, neuropeptides, and/or steroids). Otherkit embodiments comprise a solid support having a capture reagent thatspecifically binds an irrelevant molecule present in an aAPC accordingto the invention. In another embodiment, the kit may comprise virtualaAPCs on solid supports comprising a single lipid layer. Still otherembodiments employ aAPCs of the invention linked to particles that caneasily be separated from a biological sample, such as magnetic or metalparticles.

Other devices useful for isolating and modulating antigen-specific Tcells are column devices. In these embodiments, the devices may comprisecompartments that may be isolated from one another by way of separateentrance and exit flow ports between said compartments and between thecompartments and external apparatuses. One or more compartments of suchcolumn devices may further comprise solid supports capable of bindingirrelevant and other such molecules present in aAPCs or solid supportsthat function directly on aAPCs as described above. Such devices may beused in connection with soluble immunomodulatory molecules that areneither bound to a solid support nor incorporated into an aAPC. Examplesof such molecules include cytokines, chemokines, and hormones.

In another such example, if leukopheresis is being performed with theintention of reintroducing the cells back into the patient's body,soluble factors may be introduced into a column device to induceproduction of IL2 in na{dot over (i)}ve T cells, IL2 being necessary forT cell growth. Likewise, IL4 or soluble IL4 receptor antibody may beintroduced into an immunomodulatory column to enhance the maturation ofthe Th2 phenotype in specific na{dot over (i)}ve T cells of interest.

Hence, the invention further comprises a method of modulating T cellresponses (i.e., altering a T cell's phenotype, activation, and/orpriming). In such embodiments, methods of regulating or modifying T cellresponses ex vivo, such as in a column device of the invention, areprovided comprising the steps of isolating T cells which are specificfor an antigen of interest and combining said isolated T cells with anartificial antigen presenting cell. The aforementioned steps may beperformed simultaneously or separately. For instance, T cells specificfor an antigen of interest may be isolated using the T cell isolationmethods described above. The captured antigenic-specific T cells in onecompartment in the device detailed above, for example, may then beintroduced into another compartment into which an aAPC is introduced oralready present.

The modulation of such T cell responses may comprise changing, in wholeor in part, the functional pattern of cytokine receptor expression,cytokine production and secretion, chemokine production and secretion,and/or chemokine receptor expression by the isolated T cells specificfor a given antigen. One of the many reasons for modulating a givenpopulation of T cell responses may be to change, in whole or in part,the functional pattern of cytokine production by the isolated T cells.For instance, a na{dot over (i)}ve T cell may be stimulated to shift itsphenotype from a Th0 to a Th1, Th2, or a Th3 type helper T cell.Alternatively, the balance between Th1 and Th2 phenotypes may beshifted. Hence, using the methods described herein, a Th2 response maybe increased while at the same time a Th1 response may be decreased. Inthis instance, the aAPC used should also express the co-stimulatory B7-2molecule. On the other hand, a Th1 response may be increased and a Th2response decreased. In this instance, the aAPC used preferably containsthe co-stimulatory molecule B7-1.

Hence the present invention provides methods for treating variousdiseased conditions in a subject who would be benefited by modulating Tcell producation and maturity as well as the functional pattern ofactive factors expressed by such T cells. For example, as set forthabove, production of cytokines by a T cell may be modified in certainantigen-specific T cells to increase Th2 response and/or decrease Th1response. In such a method, a subject's T cells that are specific for anantigen capable of triggering a Th1 response are isolated by contactingsaid cells with an APC having an MHC:antigen complex containing anappropriate antigen. By also including a co-stimulatory molecule such asB7-2 on the aAPC, the T cells may be directed to modify their responseand cytokine production to increase their Th2 response. Conditions thatwould benefit from altering the functional pattern of response toward aTh2 response include, for example, autoimmune diseases such as type 1diabetes mellitus, multiple sclerosis, rheumatoid arthritis,dermatomyositis, juvenile rheumatoid arthritis, and uveitis.

Such T cell modulation, as described above, may be useful in thetreatment of autoimmune diseases and asthma, as well as well as variouscytokine-induced inflammatory diseases. For instance, a subject maybenefit from altering the functional pattern of cytokine production bycertain antigen-specific T cells to increase a Th1 response and/ordecrease a Th2 response. Such methods comprise isolating a subject's Tcells that are specific for an antigen capable of triggering the Th2response by contacting said cells with an aAPC containing an MHC:antigencomplex having an appropriate antigenic peptide, wherein said artificialAPC also expresses a co-stimulatory molecule such as B7-1. For example,it is known that ST2L expression is Th²-type specific. Hence, in thecurrent invention, ST2L may be included in an aAPC in order to identify,isolate, and extract antigen-specific T cells of the Th2 phenotypeand/or enrich T cell population for antigen-specific Th1 cells.Additionally, CD30, which is known to have an association with asthma(Spinozzi, F., Mol Med., vol. 1 :7:821-826.),may be incorporated into aAPCs to also identify, isolate and remove antigen-specific T cells ofthe Th2 phenotype and/or to induce T cell responses away from harmfulTh2 helper cells in the treatment of allergic conditions. Morespecifically, allergic conditions that benefit by altering thefunctional pattern of cytokine production to increase Th1 responsesand/or decrease Th2 responses including, for example, allergies to dustmites, animal skin bypass products (e.g., cat and dog dander),vegetables, fruits, pollen, and other various chemicals. Otherconditions that may benefit include cancers and some types of infections(e.g., viral, protozoan, fungal, and bacterial infections).

In another example of a method of treatment according to the invention,artificial APCs may be designed to augment antigen-specific T cellresponses away from the harmful helper type T cells or used to depleteoffending T cells in the treatment of multiple sclerosis. Such depletionor modulation of the T cells may be carried out in combination with theinfusion into a column device of either Fas, Fas Ligand, anti-Fas, oranti-Fas ligand antibody. Additionally, artificial APCs may be designedto incorporate SLAM-reactive molecules, which then function to induce asuppressive phenotype.

In another treatment example, SLAM-reactive molecules incorporated in anartificial APC may be used to treat Th2-mediated autoimmune diseases bymodulating the T cell response to shift from a Th2 profile to a Th0/Th1profile while at the same time inducing IL-2 independent co-stimulationindependent proliferation.

On the other hand, the OX40 ligand is known to induce a Th2-likephenotype in na{dot over (i)}ve T cells (Flynn, S., Journal ofExperimental Medicine, vol. 188:2:297-304). In this case the OX40protein may be incorporated into an aAPC to selectively induce a Th2phenotype.

In yet another example, cartilage degradation that is associated withrheumatoid arthritis can be treated and even in some instances preventedby use of IL-10 and IL-4 in association with an artificial APC, or byinfusion of the soluble molecules into a column device, In part, theassociated IL-10 and IL-4 molecules can help induce antigen-specificna{dot over (i)}ve T cells away from development into the activated Th1state. On the other hand, IL-12 can be used, in accordance with themethods of this invention, by infusion into a column device orincorporation in an artificial APC to induce a Th1 type inflammatoryresponse to help treat Th2 mediated autoimmune diseases.

The modulation of a T cell response may also be useful for inducinganergy and/or apoptosis. The onset of anergy is reported to be due, inpart, to the presentation of MHC bound antigenic factors for T cellactivation in the absence of appropriate co-stimulatory molecules. (Dingand Shevach, European Journal of Immunology, vol. 24:4:859-866) Hence,the instant invention is applicable to situations where anergy may beuseful in the treatment of varius maladies, such as in the induction oftolerance. For instance, various forms of Inflamatory Bowel Disease,Crohn's disease and colitis can be alleviated through tolerancemechanisms. Tolerance-inducing mechanisms may also be useful in thetreatment of various forms of allergic responses. In these regards, theaAPC of the invention will process and display an appropriate antigenicepitope or allergen but will not express an appropriate co-stimulatorymolecule, but may effectively express other effector molecules, e.g.,Fas ligands, which may help induce anergy. Thus, an aAPC used in thismethod would not express a co-stimulatory molecule, but mayalternatively express Fas ligand, to induce anergy or tolerance. Suchtolerance may be induced orally, sub-cutaneously, intraveniously, or viaother suitable routes.

In another example of tolerance induction, it is known that thecross-linking of the CD40 ligand using antibodies induces cellproliferation and IL-4 production (Blotta, M., H. J Immunol., vol.156:3133-3140). Additionally, it is known that blockage of the CD40/CD40ligand pathway induces tolerance in murine contact hypersensitivitystudies (Tang, A., European Journal of immunology, vol. 27:3143-3150).Hence, in some embodiments of the current invention, CD40 or theanti-CD40 ligand antibody may be incorporated into an artificial APC toinduce T cell modulation toward production of IL-4 and/or tolerance toalleviate inflammatory autoimmune disorders.

In other embodiments, the modulation of a T cell response may simplycomprise inducing T cell proliferation in general, without regard tomodifying Th1 or Th2 responses, and without regard for inducing anergy.This may be accomplished by use of an aAPC that expresses an anti-CD28antibody. These methods of treatment can include, in addition to the useof artificial APCs, the use of a column device described herein andabove.

In another example of a method of treatment, it is known that anti-B7-1antibody will reduce the incidence of EAE, an animal model of multiplesclerosis. Anti-B7-2 antibody is known to increase the severity of EAE,while co-treatment with anti-IL4 antibody will prevent diseaseamelioration. Hence, the current invention provides more than one levelof control with respect to this disease. For example, artificial APCsmay be generated that express anti-B7-1 antibody and/or IL4 to elicit Tcell responses favorable to treating this disease.

In another example, a regimen may be developed for treating melanoma byscreening for T cell responses to epitopes derived from MAGE-1, MAGE-3,MART-1/melan-A, gp100, tyrosinase, gp75, gp15, CDK4 and beta-catenin,all of which are known to be associated with the disease. T cells havingspecificity for these molecules may be activated and modulated invarious ways described herein in accordance with the invention. Forexample, T cells that are specific for a unique cancer related antigencan act to cause destruction of the cancerous cells and may beproliferated and infused into a patient.

In another example, T cell mediated milk intolerance as well as othervarious allergic responses may be treated by isolation and depletion ofT cells specific for those allergens (e.g., Fel, Fel-d1, Der 1 and 2,etc.) that are incorporated as an MHC bound antigen on an artificialAPC.

In still another example, T cells specific for the ras peptide, orvariants thereof, may be stimulated by response to an artificial APCcontaining the ras peptide in the treatment of cancerous cellsexpressing the ras mutation.

Another aspect of the invention is that it allows for the study and useof T cell modulation in vitro, which can then be applied in an ex vivotreatment regeime. For example, due to the fact that the bioavailabilityof injected complexes is difficult to understand (see, Gaur, A. J ofNeuroimmunol. 74:149-158.) the in vitro/ex vivo methods of the presentinvention allows one to follow the specific actions of peptides ofinterest when used either in an artificial APC or in soluble form in acolumn device. Hence, it has been shown that using I.V. injections ofthe non-encephalitogenic APL91 peptide of MBP, ameliroates disease byshifing cytokines from inducing the Th1 to the Th2 phenotype. The sametype of injections using encephalitogenic superagonist APL A97 alsoameliorates disease, but by causing deletion of specific T cells.

Prior studies have also indicated that the in vivo application ofpeptides to treat autoimmune disease states is related to epitopespreading, which results in relapsing episodes of disease (Lehmann, P.V. Nature. 358:6382:155-157.),(McRae, B. L. Journal of ExperimentalMedicine. 182:75-85.). The ex vivo application of the current inventionallows specific peptides to be used to isolate and identifyantigen-specific T cells, without exposing a patient to the danger ofepitope spreading that is associated with relapses of certain autoimmunediseases.

In a further embodiment of the invention, a method of identifying Tcells that express MHC epitopes important to graft versus host diseaseand the rejection of transplantation therapy is provided. In thisembodiment, MHCs are identified and incorporated into an artificial APC.Such APCs may be used to capture and deplete the recipient's T cellshaving antigen specificity for such MHC:epitopes, this modulation allowsa more favorable response of the recipient's immune system to the graft.More specifically, peptides derived from the MHC of a recipient arebound to the MHC of a donor that are incorporated into artificial APCs.Such APCs are used in combination with tolerogenic stimuli also on theAPC or infused into an immunomodulatory column. The donor's T cells canthen be screened to bind reactive T cells that can then be discarded. Ina preferred embodiment, the column device of the invention can be usedto carry out such immunoleukophoresis.

In another embodiment, the method contemplates treatment of anindividual to cause a favorable immune response to an allograftcomprising:

-   -   (a) predicting a donor's MHC to which a recipient may react;    -   (b) testing the predicted MHC epitopes with a recipient's T        cells (in vitro) to identify antigenic epitopes;    -   (c) using identified epitopes in an artificial APC to deplete        the recipient's antigen-specific (i.e., donor-specific) T cells,        while additionally desensitizing the recipient to the epitope by        contacting the recipient (as by feeding or nasal injection) with        increasing doses of the donor-specific antigen (for the        production of oral tolerance and anergy).

In still another embodiment, the invention provides a method ofidentifying a particular individuals' MHC epitopes that are of import inimmunologic responses to pathogenic agents. In this embodiment, anindividual's MHC is screened for epitopes that have appreciable sequenceor molecular structure recognition with pathogen-derived molecules. Theidentified MHC epitopes can be used to elicit immunity that may beemployed directly as a vaccine against such MHC epitopes that havesequence recognition with pathogen-derived peptides. For example, such avaccine may be used to reduce natural APCs that express MHC moleculesassociated with autoimmune diseases including, but not limited to,multiple sclerosis, rheumatoid arthritis, and diabetes. In anotherembodiment, the identified MHC epitopes can be incorporated into aliposome structure along with a co-stimulatory molecule to enhance theeffect of artifical APCs that express other disease related antigens.

In yet another embodiment, antigenic moieties of the pathogen whichhave, or are likely to have, MHC mimics may be used in artificial APCMHC:antigen complexes to isolate T cells specific for suchpathogen-derived antigenic motifs or mimics thereof for the productionof a T cell vaccine. Such a vaccine may be used to directly fightprogression of an infection or disease caused by a pathogen, or that isthe result of a pathogen-derived antigen induced autoimmune associatedinflammatory response. Usually a self-derived molecule that mimics apathogen-derived antigen comprises a polypeptide. As used herein, such amimic has an amino acid sequence identity with said pathogen-derivedantigen to an extent necessary for the MHC to bind to the mimic. Therange of sequence identity may be anywhere between 5 and 100% dependingupon which amino acids in a peptide sequence elicits either recognitionby the MHC and/or stimulation of a T cell response. Generally, the rangeis between about 5 and 100%, usually, the range is between of a range ofof about 15 and 100%, preferably the range is between 35 and 100%, andmost preferably the range is between 50 and 100%.

In still other embodiments, the invention provides a means to addressother immunologic conditions relevant to a T cell response. For example,apoptosis of T cells induced by MHC molecules through the CD95/CD95ligand pathway can be controlled by incorporating an anti CD95 moleculeinto an artificial APC and modulating the appropriate antigen-specific Tcells.

In other applications, dendritic cells, and other similar surveliencecells, important to an immune response, may be manipulated ex vivo inthe same fashion as T cells in the many examples provided above.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings are provided to the Patent andTrademark Office with payment of the necessary fee. The invention willbe further described with reference to the accompanying drawings, whichcan be briefly described as follows:

FIG. 1 is a schematic representation of the basic features of theinvention and it's interaction with various molecules on a T cell, where(1) is an artificial APC in which embedded molecules have conformed tothe capping of the T cell, where (2) is an artificial APC that has notinteracted with a T cell and who's molecules are randomly dispersedthrough the membrane, where (T) is a T cell who's molecules have cappedin response to the interaction with the artificial APC, where (3) is anaccessory molecule for stabilizing the binding between the TCR andMHC:antigen complex, where (4) may be T cell accessory or adhesionmolecule ligands, where (5) may be T cell co-stimulatory moleculeligands, where (6) may be an MHC:antigenic peptide complex, where (7)may be cytokine related molecules, where (8) may be chemokine relatedmolecues, where (9) may be an irrelevant molecule used in binding of theartificial APC to a solid support or a molecule which is tagged with amolecule used in visualization.

FIG. 2 is a schematic representation of an embodiment of the inventionrepresentative of a portion of an artificial APC (10) in which thepeptide (11) complexed to the MHC (12) is tagged, wherein the tag, forexample, is biotin (13). The visualization of the complex comprising theabove components may occur (while a TCR (14) on a T cell (15) is boundto the APC) by FACS through the addition of a streptavidin moleculecomplexed to a fluorochrome (16).

FIG. 3 is a schematic representation of an embodiment of the inventionrepresented by a portion of an artificial APC (17) interacting with a Tcell (18) through the TCR (19), using the artificial APC's MHC (20) andunlabeled peptide (21). The visualization by FACS is performed by theinclusion of an irrelevant molecule (22) having an attached label (23).The irrelevant molecule minus the label may also be used to bind theliposome to a solid support.

FIG. 4 is a schematic representation of an embodiment of the inventionrepresented by a portion of an artificial APC (24) interacting with a Tcell (25) through the TCR (26), using the artificial APC's MHC (27) andunlabeled peptide (28). A fluorochrome (29) may be added to the dialysisbuffer during the formation of the liposomes so that visualization maybe carried out by FACS.

FIG. 5 is a schematic representation of an embodiment wherein theartificial APC (30) includes a functional molecule (31) (such as anaccessory, co-stimulatory, adhesion, modulation, cytokine, or chemokinemolecule) that interacts with a molecule (33) expressed by a T cell 32.

FIG. 6 is a schematic showing the capture of artificial APCs (35) and(37) by capture molecules (39) that are noncovalently associated with alipid layer (38) (e.g., a neutral phospholipid) attached to a solidsupport (34).

FIG. 7 is a schematic showing other embodiments of artificial APCdesigns, wherein FIG. 7 a represents a solid support (40) having anon-covalently associated lipid layer (42) containing various componentsof artificial APCs (41) that can bind a T cell (43). FIG. 7 b shows asolid support (44) having the various components of artificial APCs (45)without a lipid component that can bind to a T cell (46).

FIG. 8 shows a column device having a multiplicity of compartments forprocessing antigen-specific T cells. The column, as shown, also containsaAPCs and various embodiments of solid supports.

FIGS. 9A and B is a series of FACS figures detailing the specificity ofthe embodiment of the method diagramed in FIG. 2 using two differentmurine hybridomas specific for the OVA peptide in the context of eitherIα52 or I-A^(s). The Hi15 peptide has two identities and oneconservative substitution with the OVA peptide, thus being a stringentcontrol of specificity.

FIGS. 10A, B, and, C show a series of experiments from a non-transgenicmurine model in which the characterization of the antigen specificity(the Iα52 peptide) of a T cell population without the use of the currentinvention was attempted.

FIG. 11 shows the ability of the invention to determine directly theantigen specificity of a T cell population in the non-transgenic mousemodel that could only be inferred in FIG. 10.

FIG. 12 shows the ability of the invention to determine thecross-reactivity of a singular group of antigen-specifically defined Tcells seen first in FIG. 10, then in FIG. 11. Such a techniquerepresents an improvement over prior art, as internalized antigen cannot be removed.

FIGS. 13A-E. is a series of FACS figures showing the use of theinvention to identify T cells based on the specificity of the TCR in thePBMC of a patient with RA.

FIGS. 14A-D is a series of FACS figures showing the use of the inventionto identify T cells based on the specificity of the TCR in the PBMC of apatient with JDM.

FIG. 15 shows the identification of antigen specific cells with T cellcapture in a monoclonal TCR population wherein OVA³²³⁻³³⁹/IA^(d)specific T cell hybridoma 8D0 cells were incubated with artificial APCscomplexed with IA^(d) and biotinylated OVA³²³⁻³³⁹. Prior to theincubation, the hybridoma cells were stained with CD4 PE (phycoerithrin,i.e., a fluorochrome) and the artificial APCs, complexed with IA^(d) andbiotinylated OVA³²³⁻³³⁹, were stained with streptavidin labeled with CY.As control for specificity of the binding, T cells of the same hybridomawere incubated with liposomes complexed with IE^(d) and biotinylatedOVA³²³⁻³³⁹. Binding of hybridoma T cells to artificial APCs couldbeinhibited by co-incubation with monoclonal anti IA^(d) antibody. Thehistograms show the intensity for staining for streptavidin CY in CD4gated hybridoma cells. Gates were set on irrelevant isotype controls forCD4, and on binding of cychrome-conjugated streptavidin to Tcells/artificial APC with unbiotinylated OVA³²³⁻³³⁹ peptide.

FIGS. 16(A-D), 17(A-D), and 18(A-D) are color photos showing that theinteraction of T cells with artificial APCs allows physiologicalmigration of TCR proteins toward the interaction side, i.e., “capping”.Resting 8DO T cells were stained with FITC-cholera toxin, incubated withartificial APCs presenting IAd^(/)OVA³²³⁻³³⁹ complexes and analyzed byconfocal microscopy. Panels A and B of each of FIGS. 16-18 show the redand green fluorescent dyes, respectively. Panel C shows the cells asseen by phase contrast. Panel D is the combination of the twofluorescent dyes with phase contrast. Panel D shows the combinedfluorescence of the red and green dyes so that co-localization (FIG.16D), and capping (FIGS. 17D, and 18D) are observed. Specifically, FIGS.16A-D shows 8DO cells alone stained with Alexa 568-anti CD3 (red) boundto the T cell receptor and FITC-cholera toxin (green) bound to thecholera toxin. Thus, FIGS. 16A-D shows that the T cell receptor isassociated with the cholera toxin antigen. This further shows thatcholera toxin is a good tool for identifying the T cell receptor in thissystem. FIGS. 17A-D shows 8DO cells incubated for 20 minutes withartificial APCs expressing IA^(d)/OVA³²³⁻³³⁹ complexes. Artificial APCswere stained with Alexa568 anti MHC (red) (i.e., the MHC is labeledred), and FITC cholera toxin (green) (i.e., the cholera toxin bound tothe T cell receptor is labeled green). Thus, FIGS. 17A-D shows that theinteraction between T cells and artificial APCs allows physiologicmigration and capping of the T cell receptor in the T cell membrane.FIGS. 18A-D shows 8DO cells incubated for 20 minutes with artificialAPCs expressing IA^(d)/OVA³²³⁻³³⁹ complexes. Artificial APCs lipidmembranes were labeled using FITC (green). The T cell receptor werelabeled with Alexa568 anti CD3 (red). Thus, FIGS. 18A-D further confirmsthe data shown in FIGS. 17A-D.

FIGS. 19A-D are PAGE photos showing optimization of peptide (antigen)loading of human and mouse MHC class II molecules wherein detergentsolubilized MHC class II molecules were incubated with biotinylatedpeptides at the designated pH and temperature for 16 and 24 hrs. Peptideloading was analyzed through ECL. FIGS. 19A and B show binding ofbiotinylated PADRE peptide to HLA-DR4. FIGS. 19D and C show binding ofbiotinylated OVA³²³⁻³³⁹ to mouse IA^(d). The photos show output signalof the level of binding of the peptides to MHC. Results indicate that anincubation of 16hrs at room temperature and pH 7 and a molar ratio of200 to 1 provide for optimal results with respect to the human MHC and1000 to 1 for the mouse.

FIG. 20 is a graph showing characterization of the artificial APCs byflow cytometric analysis wherein the approximate size of thefluorescent-labeled liposomes was determined through comparison withsingle size fluorescent particles with a mean size between 0.05 um and0.85 um. The 0.85 um labeled curve represents beads of 0.85 um diameter,while the curve labeled 0.05 represents beads of 0.05 um in diameter,the shaded curve represents the size of APC.

FIG. 21 is a graph showing determination of the optimum incorporation ofMHC class II in liposomes. Optimum incorporation is a factor of theratio of the phospholipid and cholesterol. The ratio of the lipidcomponents were tested such that fluorescent labeled liposomes werecomplexed with HLA DR4 where the ratio was from 3.5:1 (w/w) to 14:1 andstained with anti-HLA DR PE. The incorporation of DR4 was measured bymeans of flow cytometric analysis of the signal for HLA DR-PE in FL2(X-axis). As shown the number of incorporating events is highest (xaxis) at a lipid/cholesterol ratio of 7:1.

FIG. 22 is a graph showing incorporation of biotinylated PADRE influorescent labeled liposomes, complexed with HLA DR4 and specificity ofthe binding of biotinylated PADRE peptide to HLA DR4 complexed withliposomes. Biotinylated PADRE peptide is incubated with HLA DR4 or MHCClass I, both at a molar ratio of 10:1 (peptide to MHC) prior toincorporation in artificial APCs with fluorescent lipids. As anadditional control, non-biotinylated PADRE peptide and biotinylatedPADRE peptide (at a 1:1 w/w ratio) were incubated with HLA DR4 beforeincorporation in artificial APCs. The PADRE is a pan DR binding peptide.The graph indicates specificity and that label bound to the PADRE doesnot interfer with binding to the MHC component. Specifically, wherenon-biotinylated peptide is used at the same time, it will compete outbiotinylated. The Class I binding shows that there must be specificityfor such binding as use of the Class I fails to bind PADRE.

FIGS. 23A-F show plots of FACS analysis showing that T cell capture isan effective method to identify class II restricted human polyclonal TCells. The panels show a comparison of CD3+ cells binding PADRE/HLA orHA/HLA (HA is hemoglutinin A) complexes. PADRE is used as positivecontrol because it will bind many MHC molecular species therebyproviding an easy means to visualize populations of specific cells. HAis also a pan DR binding peptide but because cells in this example areexpanded using PADRE peptide, use of HA for binding should serve as anegative control. The Y axis represents CD3+ cells. The x axisrepresents HLA/PADRE or HLA/HA complexes. Panels A and B show the % ofPADRE antigen specific T cells at day 0 of culture (A) (i.e., 2.9%), andthe % of antigen-specific T cells at day 10 of culture with PADRE (B)(i.e., 8.1%). Thus, cells appear to be expanding as an antigen specificfashion. To test the specificity of the cell population, the % of HAcells are tested. As shown in panels C and D respectively, the % of HAspecific T cells after 0 days of culture with PADRE/HA (C) (i.e. 1.0%)drops to 0.3% HA specific T cells after 10 days of culture with PADRE(D). Panels E and F show the % of antigen specific T cells at day 10 ofculture wherein T cell capture using aAPCs was inhibited with anequimolar ratio of non-biotynilated PADRE. Panel E shows that 50% ofinhibition was achieved, (i.e., the label does not interfer with testingof specificity using the APC). Panel F shows that 50% of inhibition wasalso achieved showing that the T cell/aAPC binding depends on thespecific interaction of the T cell receptor, i.e., inhibition of bindingusing anti-HLA DR antibody at a molar ratio of 0.5:1 antibody to HLA.The FACS plots show results from an experiment wherein PBMC from aHLA-DR4 0401 + donor were stimulated with 10 ug/ml of PADRE peptide,K(X)VAAWTLKAA (Seq. Id. No. 7) where X is a derivatized amino acid suchas cyclohexylalanine. At days 4 and 7,10 ng/ml IL-2 was added. At day 10cells were harvested for T Cell capture. HLADR4 was complexed withNBD-labeled liposomes (1:7 ratio HLA to liposomes) through 48 hoursdialysis against PBS in 10.000 M cutoff dialysis membrane (Pierce).Complexes were then incubated for 48 hours at RT with n-terminusbiotinylated peptides at a molar ratio of 10:1 for peptide/HLA. Excessof unbound peptide was removed through 24 hours of dialysis against PBS.Liposome-HLA-peptide complexes were incubated for 30 minutes withstreptavidin-Cy before adding to the cells. The liposome-HLA-peptidecomplexes were then incubated with the stained cells and run on a BectonDickinson FACS Star. Gates were set on viable cells, isotype controlsand cells incubated with streptavidin CY alone.

FIG. 24 is a bar graph showing cytokine production by PADRE-stimulatedcells. Production of IL-2 and IFN in vitro of PBMC from HLA-DR 0401healthy adults. PBMCs were initially stimulated with 10 ug/ml of Pan DREpitope binding peptide (PADRE) and cultured for 10 days. The cells wererestimulated with autologous APCs and 10 ug/ml of peptide. Culturesupernatants were collected at different days and measured for cytokineproduction by capture ELI SA method. The result indicates that cellspecificity can be shown by measuring cytokine production as cellsproliferate. However, measuring cytokine production is actually lessspecific than the method of the invention as demonstrated in FIG. 23.

FIGS. 25A and B are FACS plots showing identification by T cell captureusing artificial APCs of class II-restricted antigen specific mouse Tcells upon immunization and shows increase of IA^(d)/Ia52 specific cellsmeasured after immunization of the mice (at the base of the tail) withla52 or IFA (adjuvant) alone. Cells were harvested three days after thelast immunization from inguinal draining lymphnodes, stained withanti-mouse CD4 PE and incubated with fluorescein labeled artificial APCscomplexed with IA^(d)/Ia52. FIG. 25A shows IFA only-immunized mice; FIG.25B shows IA^(d)/Ia52 specific CD4 cells. Y axis: CD4; X axis:IA^(d)/Ia52 specific T cells. The result indicates that the adjuvantimmunized cells showed only 0.7% specific cells whereas the IA52immunized cells comprised 5.4%. Thus, T cell capture using artificialAPCs is useful to show antigen specificity.

FIG. 26 is a bar graph showing T cell proliferation to Ia52 afterimmunization. Cells were harvested from inguinal lymphnodes andincubated for three days with 10 mg/ml of Ia52 peptide. Proliferationwas measured by thymidine incorporation and is expressed as stimulationindex “SI”: cpm of stimulated/unstimulated cultures. The resultindicates that conducting T cell proliferation tests to determinespecificity is effective in measuring the specificity of T cellpopulations. However, the specificity is not to the same extent as thatusing the current invention shown in FIG. 25.

FIGS. 27A-C is a schematic showing methodology for orienting moleculesof interest in the APC liposome matrix. In 27A, a molecule of interestsuch as MHC, functional, accessory, adhesion, or irrelevant molecule,may be synthesized by recombinant methods well known to those skilled inthe art and linked to GM-1 by a linker and properly oriented in the APCmembrane. In 27B, a molecule of interest may be constructed as a fusionprotein with cholera toxin β subunit and the fusion protein anchored inproper orientation in the APC membrane by the cholera toxin moietybinding to GM-1. In 27C, a cholera toxin subunit may be chemicallylinked to SPDP linker (Pierce) and then attached to a molecule ofinterest followed by anchoring to a GM-1 containing aAPC. In any of theabove, the cholera toxin, GM-1, linkers may be synthetically produced.In the figure, A represents a gene for a molecule of interest, Brepresents the gene for the i subunit of cholera toxin, A1 is anexpression vector, A2 represents expression and isolation of the clonedgene, A3 is an expressed molecule of interest, B I is a fusion proteinof a molecule of interest and cholera toxin, A4 is a linker, A5 is anartificial APC containing GM-1 protein, A7 is a partial view of anartificial APC wherein the molecule of interest is directly linked tothe GM-1, C is choler toxin subunit, C1 is a linker, C2 is a molecule ofinterest, C3 is a choler toxin subunit attached to a linker, C4 is amolecule of interest linked to a choler toxin subunit, E represents aliposome bilayer, E1 shows GM-1 molecules, E2 is an artificial APCcontaining GM-1, and F is a partial view of an artificial APC having amolecule of interest bound to the APC by the binding interaction of theGM-1 and cholera moiety.

FIG. 28 shows a diagram wherein aAPC lipid bilayer 100 contains apentameric GM-1 raft 101 that is bound to five cholera toxin β subunits102-106 (each unit of the pentamer bound to a single toxin subunit). Asindicated, each of 20 the toxin subunits comprise a fusion of a completecholera β subunit and an immunologically active protein sequence ofinterest 108-112 connected through a linking sequence 107.

FIG. 29 shows DNA (Seq. Id. No. 15) and its amino acid translationsequence (Seq. Id. No. 16) wherein the DNA sequence encodes B7-1 fromits 5′ initiation codon to its 3′ codon for the carboxy terminal Serinewhich is connected to a sequence encoding a spacer/linker peptide, whichis connected in turn by a nucleic acid sequence encoding the full lengthmature cholera toxin β subunit. Sequence for the linker section isunderlined.

FIG. 30 shows a DNA sequence (Seq. Id. No. 17) and its amino acidtranslation sequence (Seq. Id. No. 18) comprising a fusion proteinencoding B7-2 from its initiation codon to its 3′ codon for the carboxyterminal Histidine followed by a sequence encoding a spacer/linkerpeptide, followed in turn by a nucleic acid sequence encoding the fulllength mature cholera toxin β subunit. Sequence for the linker isunderlined.

FIG. 31 shows the DNA sequence (Seq. Id. No. 21) and its translatedreading frame for the encoded amino acid sequence (Seq. Id. No.22) ofthe α domain of HLA DRB 1 *0401 (comprising α1 to α2) fused to a linkersequence, a protein binding Leucine zipper A, followed in turn by asecond linker sequence and the cholera toxin β subunit.

FIG. 32 shows the DNA sequence construct (Seq. Id. No. 23) and aminoacid sequence translation (Seq. Id. No. 24) of the HLA DRB1 *0401 βdomain (comprising β1 to β2) fusion with a linker sequence, followed byLeucine zipper B, followed in turn by a second linker sequence, which isfollowed by a biotag peptide sequence (and an irrelevant molecule).

FIG. 33 is a schematic showing a pentameric aggregation of GM-1 bindingto five cholera toxin subunits 120. Each cholera toxin β subunitcomprises a fusion protein wherein the toxin moiety is connected to alinker 121, followed by a Leucine zipper A sequence 122, followed inturn by HLA DRB1*0401 α123. The Leucine zipper A 122 is bound to theLeucine zipper B component 124 of a second fusion protein constructcomprising the β domains of HLA DRB1 *0401 125 which is fused to alinker, followed by the Leucine zipper B 124, followed in turn byanother linker 126 and finally to a biotag moiety 127. Also, depicted isantigen peptide 128 bound to the HLA α and β moieties, as well asaccessory fusion constructs 129 depicting the variable nature of theGM-1 ganglioside-based rafts of the invention.

FIG. 34 depicts a GM-1 molecule 130 bound by a cholera toxin molecule131 that has been biotinylated 132 at its 5′ end. The biotin is bound toone of the binding centers of neutravidin 133 that is in turn boundthrough its remaining three binding centers 134 to any immunologicallyactive molecule of interest. Specifically depicted are binding to anaccessory molecule 135, MHC with its antigen peptide 136, and anti-CD 28antibody 137.

FIGS. 35A and B shows FACS analysis wherein GM-1 rafts in liposomes areshown to bind to cholera toxin-conjugated with FITC label.

FIG. 36 shows a saturation curve wherein increasing amounts of choleratoxin is added to a fixed GM-1 population (approximately 6,374 picomoles) on the aAPC. FACS analysis visualizes cholera toxin bound on theaAPC showing saturation is obtained by addition of at least 5,000picomoles of the toxin. The table shows GEO, which represents the meanfluorescent intensity observed for each condition which translates intothe % of aAPCs containing toxin, the percent saturation expressed by the% gated M2 column.

FIG. 37 shows binding of GM-1 raft based aAPC containing HLA, HA andanti-CD28 vs. non-GM-1 raft based liposome containing randomlydistributed HLA and HA. Identification of HA-positive CD4+ cells wassignificantly more efficient when both biotinylated HLA/HA and anti CD28molecules were present on the micromembrane domains on the aAPC(17.8+5.2% CD4+ cells. In comparison, only a small proportion of antigenspecific T cells was visualized when only HLA/HA complexes were deployedon the aAPC using non-GM-1 based rafts for visualization of the antigenspecific T cells. Only 2.4+1.6% of CD4+ cells was bound by aAPC in whichHLA/peptide complexes were randomly distributed on the lipid bilayer aspreviously described.

FIG. 38 shows relative binding of CD 4+T cells by GM-1 raft based aAPCcontaining HLA and HA and anti CD28 vs. non-GM-1 raft lipsomescontaining HLA and HA. As indicated, the GM-1 based aAPC of the currentinvention exhibited approximately an eight fold increase in the bindingefficiency.

FIGS. 39A and B show efficiency of activation of IL-2 production in CD4+cells by FACS analysis using either GM-1 based rafts containing anti-CD3and anti-CD28, or a non-GM-1 raft system (e.g., a planar array format).FIG. 39A shows that in the non-raft system only 7% of cells werestimulated as opposed to the raft system (FIG. 39B) wherein 16.2% of thecells were stimulated.

FIG. 40 shows that CD4+T cells can be modulated to express CD69 using ofa combination of anti-CD3 and anti-CD28. This combination provides asignificantly higher stimulation of expression of CD 69 than when eitheranti-CD3 or CD28 are used in the aAPC alone.

FIG. 41 shows IL-2 production by CD4+ cells following stimulation withGM-1 based aAPC. As indicated, use of a combination of anti-CD3 andanti-CD28 provides stimulation of significantly higher IL-2 production.

FIG. 42 shows specific activation of CD4+ cells to express CD69.Particularly, aAPC of the current invention provide a three-foldincrease in stimulation over liposome constructs not containing GM-1based rafts.

FIG. 43 shows specific activation of CD4+ cells to express IL-2.Particularly, aAPC of the current invention provide almost a three-foldstimulation over liposome constructs not containing GM-1 based rafts.

DETAILED DESCRIPTION OF THE INVENTION

The immunoregulation art has advanced steadily in recent years. Thescientific literature contains many studies reporting interactions andmodulation effects between specific molecules and cell types. However,no discovery has been presented that is able to apply the knowledge thathas been gained by the extensive research in the field toward methods,compositions, or devices that can be used in a comprehensive package forcarrying out the identification, isolation, and modulation ofimmunoregulatory cells for the purpose of advancing the ultimate goal ofsuch knowledge, i.e, improved treatment regimens for various states ofdisease.

Here, the invention concerns a platform technology for advancingtreatment regimens requiring the immunoregulation of immune cells thatcenters around the use of an artificial antigen presenting cell (aAPC).This platform technology may be designed or programmed on demand for usein the treatment of a broad spectrum of specific disease states.Moreover, this system is versatile and applicable to all situationswhere the isolation, identification, and modulation of T cells is ofclinical import. The invention is based on the recognition of therelevance of several types of molecular entities to thestimulation/activation, modulation, and response of T cells in theirrole within the immune system, which molecules can be used to produceartificial APCs. Such artificial APCs can then be used, for example, tocapture and/or manipulate antigen-specific T cells in vitro, and well asto therapeutically or prophylactically treat diseases in vivo or exvivo. For example, they can be used in vivo to modulate T cell activity.

Historically, programming and using T cells therapeutically has beenhampered by the problem of finding a means by which the cells can behandled for such manipulation and observation of the effectiveness ofthe manipulation applied. This invention solves this problem by adoptingthe theory that a T cell can best be manipulated by using APC-likestructures and incorporating into such structures molecules that (1)bind the “artificial” APC to specific T cell types and (2) stimulate ormodulate only specifically bound T cells for any desired response. Forin vitro applications, if desired, the artificial APCs can be bound to asolid support.

Prior to the instant invention, no comprehensive system has beendisclosed, nor was it obvious that such a system would function asdesired in a universally applicable manner to activate and modulate Tcells.

Accordingly, the present invention is directed to novel aAPCs,compositions comprising these aAPCs, as well as various methods,including methods of making the aAPCs of the invention and methods ofisolating T cells specific for particular antigens of interest, andmodulating T cell function ex vivo or in vitro. Methods for modulating Tcell function in vivo are also provided. Among the methods of theinvention are those for treating conditions that would benefit from themodulation of T cell responses, for example, transplantation therapies,autoimmune disorders, allergies, cancers and viral infections, andvirtually any T cell mediated disease. The present invention is furtherdirected to T cell modulation column devices as well as kits forisolating and modulating antigen-specific T cell populations.

Artificial APC Compositions

According to preferred embodiments of this invention, an artificial APCmay comprise MHC:antigen complexes, accessory molecules, and otherfunctional molecules including, but not limited to, co-stimulatorymolecules, adhesion molecules, modulation molecules, irrelevantmolecules, anchoring components such as GM-1 ganglioside molecules andsubunits of cholera toxin attached to any of the aforementionedmolecules, and labels, some or all of which may collectively assembledinto functional aAPCs. In certain preferred embodiments, the abovecomponents are incorporated within or are associated with the lipids ofa liposome or other membrane-based vesicle such as depicted in FIG. 1.Such incorporated or associated molecules may also include GPI anchoredproteins. In other embodiments, these components are attached to anunderlying scaffold such as a dendrimer or a particle.

In preferred embodiments of liposome-based aAPCs, the lipid membranecomponent comprises neutral phospholipids such as phosphotidylcholineand surfactant elements such as cholesterol. These materials areprovided in a ratio that allows the other molecules of interest tofreely migrate, or diffuse laterally, in the membrane layer. Other lipidmembrane components include anchoring moieties such as GM-1 gangliosidemolecules, which is a transmembrane pentasaccharide protein andassociates in part with nonpolar regions of the liposome matrix. GM-1ganglioside molecules can be used in association with proteins such ascholera toxin β subunits to orient molecules of interest in the liposomematrix.

Accessory molecules may be included for the purpose of stabilizing theinteraction between a TCR and an MHC:antigen complex. Suitable accessorymolecules may include, but are not limited to, LFA-1,CD49^(d)/29(VLA-4), CD11a/18, CD54(ICAM-1), and CD106(VCAM) andantibodies to their ligands. In a preferred embodiment, the artificialAPC includes ICAM-1 as such an accessory molecule.

Co-stimulatory molecules may be included for the purpose of stimulatingor activating a TCR. Suitable co-stimulatory molecules may include, butare not limited to, B7-1, B7-2, CD5, CD9, CD2, CD40, and antibodies totheir ligands such as anti-CD28.

Adhesion molecules may be included for the purpose of enhancing thebinding association between the artificial APC and a T cell. Suitableadhesion molecules may include, but are not limited to, members of theICAM family such as ICAM 1, ICAM 2 and GlyCAM-1, as well as CD 34,anti-LFA-1, anti-B7, and chemokines such as CXCR4 and CCR5, andantibodies to selectins L, E, and P.

Modulation molecules may be included for the purpose of modulating thephenotype of a T cell. Suitable modulation molecules may include, butare not limited to, CD72, CD22, and CD58 and antibodies to theirligands, antibodies to cytokine or chemokine receptors or smallmolecules that mimic the actions of the various cytokines orneuropeptides.

Irrelevant molecules may be included for the purpose of either carryinga label or serving as a scaffold for binding to a solid support. Such amolecule can be any peptide or other molecule having characteristicsthat make it suitable for use with a liposome and antigen carrier. Suchmolecule should not interfere with the binding of a T cell to theartificial APC.

With respect to the incorporation of each of the aforementioned MHC,accessory, co-stimulatory, adhesion, modulation, and irrelevantmolecules in the artificial APC, proper orientation of these molecule'sactive centers may be provided by combining the molecules with, forexample, the 1 subunit of cholera toxin so that the cholera toxinsubunit can be recognized and bound by GM-1 ganglioside moleculesincorporated into the liposome membrane matrix. The incorporation of thecholera toxin and orientation mechanism markedly increases the abilityof the artificial APC to interact with T cells and other cells andmolecules due to the proper orientation of incorporated molecules fromabout 50% without such orienting to 90% or more with such orienting.

In preferred embodiments, the aforementioned molecules of interest maybe produced by recombinant technology as is well known to those skilledin the art. Use of recombinantly produced molecules further provides theopportunity to produce such molecules as fusion molecules comprising themolecule of interest attached to the β subunit of cholera toxin. Inanother embodiment, the recombinantly produced (or for that matter apurified natural molecule) may be linked to cholera toxin by acommercial linker.

In still other embodiments, an aAPC of the invention comprises one ormore labels, wherein the label(s) is (are) associated with at least oneof the group selected from the group consisting of a lipid bilayer ofthe liposome components, a lipid of the liposome, an antigen, an MHCmolecule, a co-stimulatory molecule, an adhesion molecule, a cellmodulation molecule, a GM-1 ganglioside molecule, cholera toxin βsubunit, an irrelevant molecule, and an accessory molecule.

Artificial APC Formation

The MHC:antigenic peptide complexes of the aAPCs of the inventioncomprise at least two components: an antigenic peptide or otherantigenic sequence with the relevant effect on the immune system; and atleast a portion of a subunit of a MHC-encoded protein involved inantigen presentation. The association between the peptide antigen andthe antigen binding sites of the MHC subunit(s) can be by covalent ornon-covalent bonding.

In other embodiments, the aAPCs of the invention may contain a compoundthat is toxic, or which can become toxic upon activation (e.g., aprodrug). Toxic agents include naturally occurring toxins,chemotherapeutic agents, and radioactive compounds. For example, anumber of protein toxins are well known in the art including ricin,diphtheria, gelonin, Pseudomonas toxin, and abrin. Chemotherapeuticagents include, for example, doxorubicin, daunorubicin, methotrexate,cytotoxin, and anti-sense RNA. Antibiotics can also be used. Inaddition, radioisotopes such as yttrium-90, phosphorus-32, lead-212,iodine-131, or palladium-109 can be used. The emitted radiation destroysthe target T-cells. In vesicle-based embodiments, the toxic compound maybe carried inside the vesicle, and is released into a T cell uponmembrane fusion. In other embodiments, the compound may be conjugated tothe scaffold or another component of the aAPC.

Each of the components of the aAPCs of the invention is described below,followed by a description of some of the preferred methods by whichthese aAPCs can be prepared and employed.

MHC-Derived Components

The glycoproteins encoded by the MHC have been extensively studied inhuman and murine systems. In general, there are two classes: class Iglycoproteins, found on the surfaces of all cells and primarilyrecognized by cytotoxic T cells; and class II molecules, which are foundon the surfaces of several cell types, including accessory cells such asmacrophages, and are involved in presentation of antigens to helper Tcells. Many of the histocompatibility proteins have been isolated andcharacterized in the scientific literature.

The Class I MHC in humans has three loci, HLA-A, HLA-B, and HLA-C, allof which reside on chromosome 6. HLA-A and HLA-B have a large number ofalleles encoding alloantigens. Class I molecules consist of a 44 kD(kilodalton) heavy chain subunit and a 12 kD B2-microglobulin subunitwhich is common to all antigenic specificities. Isolation of thesedetergent-soluble HLA molecules has been described. See, e.g., Springer,T. A., et al., Proc. Natl. Acad. Sci. USA (1976), vol. 73:2481-2485; andClementson, K. J., et al., in “Membrane Proteins” Azzi, A., ed.

Further work has resulted in a detailed picture of class Ithree-dimensional structure. See Bjorkman, P. J., et al., Nature (1987),vol. 329:506-518. It is believed that the B2-microglobulin protein andalpha-3 domain of the heavy chain are associated, while the alpha-1 andalpha-2 domains of the heavy chain form antigen-binding sites. Science(1987), vol. 238:613-614.

Class II glycoproteins have a general domain structure similar to thatof class I molecules. The antigen binding sites of class II MHC arelocated on the N-terminal domains of the two subunits, the alpha andbeta chains. A separate antigen binding site is located on each subunit.Luescher, et al., J. Biol. Chem. (1990), vol. 265:11177-11184. The alphaand beta subunits are held together by non-covalent forces. Each chaincontains two globular domains, designated alpha-1, alpha-2, beta-1,beta-2, respectively. Except for alpha-1, each of these domains isstabilized by disulfide bonds.

MHC glycoproteins (or portions thereof) can be isolated from appropriatecells or can be recombinantly produced. Methods for purifying class IIproteins are well known. (see, e.g., Turkewitz, A. P., et al., MolecularImmunology (1983), vol. 20:1139-1147), as are methods for cloning thegenes encoding these proteins (or their functional domains). Herein, anMHC molecule refers to entire molecule (such as it occurs in nature), aswell as functional subunits or domains thereof (e.g., an alpha or betachain of MHC II, a heavy chain of MHC I, the extracellular domains ofsuch subunits, etc.). In any event, a functional subunit comprising anantigen binding site and sequences necessary for recognition by theappropriate TCR. Typically, they comprise at least about 50-80%,preferably 90-95%, of the amino acid sequence of the fill-lengthprotein. Again, MHC molecules may be recombinantly produced or beisolated from the membranes of a suitable cell type. Notwithstanding theforegoing, it is well known that native forms of “mature” MHC proteinsand subunits vary somewhat in length because of deletions,substitutions, and insertions or additions of one or more amino acidresidues in the sequences. Thus, MHC components are subject tosubstantial natural modification, yet are still capable of retainingtheir respective activities. Modified MHC molecules can also be readilydesigned and manufactured utilizing various well known recombinant DNAtechniques. For example, MHC molecules can vary from naturally occurringsequences at the primary structure level (i.e., amino acid sequence) dueto the substitution, addition, deletion, and the like of one or moreamino acid residues. These modifications can be used in a number ofcombinations to produce the final modified protein chain.

In general, modifications to genes encoding MHC molecules can be readilyaccomplished by a variety of well-known techniques, such assite-directed mutagenesis (see, Gillman and Smith, Gene (1979), vol.8:81-97; Roberts, S. et al., Nature (1987), vol. 328:731-734). While theeffect of many mutations is difficult to predict, their effect can beevaluated by routine screening in a suitable assay for the desiredcharacteristic. For instance, a change in the immunological character ofan MHC molecule can be detected by competitive immunoassay with anappropriate antibody. Similarly, the effect of a mutation in the bindingsite for an antigenic peptide may be evaluated by testing the modifiedMHC molecule's ability to bind a particular antigenic peptide.Modifications of other properties can also be assayed by standardtechniques.

This invention provides amino acid sequence variants of MHC moleculesengineered for different reasons, for example, to alter the affinity ofthe molecule for antigenic peptides and/or T cell receptors, tofacilitate stability, and to ease purification of the molecules. Themodified MHCs can result in aAPCs with different plasma half lives,improved therapeutic efficacy, and fewer side effects. Preferably, thevariants will exhibit at least the same, and preferably enhanced,biological activity (for example, antigenic peptide binding) as comparedto the naturally occurring analogue. However, the variants andderivatives that are not capable of binding to their ligands may beuseful nonetheless, for example, as reagents for purifying anti-MHCantibodies from antisera or hybridoma culture supernatants and asimmunogens for raising antibodies to MHC.

Insertional variants of the present invention are those in which one ormore amino acid residues are introduced into a predetermined site in theMHC subunit and which displace the preexisting residues. For instance,insertional variants can be fusions with heterologous proteins orpolypeptides. Substitutions involve the replacement of one amino acidtype with a different amino acid type (including amino acids and aminoacid derivatives not normally found in native proteins) at a particularamino acid position in a protein. Deletional variants are characterizedby the removal of one or more amino acid residues from a MHC protein (orportion thereof). In certain preferred embodiments, the transmembraneand cytoplasmic domains of MHC molecules are deleted. This facilitatesnot only recombinant production, but also provides polypeptides that arepreferred for use in aAPC embodiments where it is not essential for apart of an MHC molecule to reside in a lipid bilayer, for example, whena biotinylated MHC molecule is associated via a liposome through amembrane-anchored complex that presents one or more streptavidinmoieties above the exterior surface of the membrane, or, alternatively,when a dendrimer is used as the scaffold for making the aAPC.

Other protein modifications can also be made. For example, MHC proteinshaving different patterns of glycosylation as compared to native MHCmolecules can be synthesized, including variants completely lacking inglycosylation (unglycosylated) and variants having at least one lessglycosylated site than the native form (deglycosylated) as well asvariants in which the glycosylation has been changed. For example,substitutional or deletional mutagenesis can be employed to eliminate,add, or replace N— or O-linked glycosylation sites. Glycosylationvariants can also be made by using different host cells. Yeast, forexample, glycosylate proteins significantly differently than mammaliansystems. Similarly, mammalian cells of a different species (e.g.,hamster, murine, insect, porcine, bovine, or ovine) or tissue origin(e.g., lung, liver, lymphoid, mesenchymal, or epidermal) than theoriginal MHC source can provide for variant glycosylation, ascharacterized, for example, by elevated levels of mannose or variantratios of mannose, fucose, sialic acid, and other sugars. In vitroprocessing e.g., neuraminidase digestion, may also be employed to alterglycosylation.

MHC molecules can be isolated from MHC-bearing cell types using anysuitable technique. Suitable techniques include solubilization bytreatment with papain, by treatment with 3M KCl, and by treatment withdetergent. Isolation of individual MHC subunits is easily achieved usingstandard techniques. For instance, in the case of class I molecules, theheavy chain can be separated using SDS/PAGE and electroelution of theheavy chain from the gel (see, e.g., Hunkapiller, et al. (1983), Methodsin Enzymol., vol. 91:227-236). Separate alpha and beta subunits of MHCII molecules can also be isolated using SDS/PAGE followed byelectroelution, as described, for example, by Gorga, et al. (J. Biol.Chem. (1987), vol. 262:16087-16094) and Dornmair, et al. (Cold SpringHarbor Symp. Quant. Biol. (1989), vol. 54:409-416). Other standardtechniques for separating molecules can also be used, such as ionexchange chromatography, size exclusion chromatography, and affinitychromatography.

Alternatively, as the genes and amino acid sequences many class I and IIproteins are known (or can be determined using techniques known in theart), MHC molecules useful in practicing the invention can also besynthesized using recombinant methods. These techniques enable numerousmodifications to MHC molecules, e.g., site directed mutagenesis, fusionwith other proteins (or protein domains), addition of amino acidresidues to facilitate purification, deletion of transmembrane andcytoplasmic domains, etc. Typically, a gene encoding a particular MHCpolypeptide will include restriction sites to aid insertion intoexpression vectors and manipulation of the gene sequence. Genes encodinga desired protein are then inserted into suitable expression vectors,expressed in an appropriate host, such as E. coli, yeast, CHO or othermammalian cell line, and insect cells. Cell-free expression systems canalso be employed. Indeed, if the desired protein is less than about 100amino acids in length, synthetic chemistry methods may be employed inits synthesis.

Construction of expression vectors and recombinant production from theappropriate nucleic acid sequences are well known in the art. Suchtechniques and various other techniques are generally performedaccording to Sambrook, et al., Molecular Cloning—A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. DesiredMHC molecules from recombinant sources are recovered and preferablypurified prior to further use.

Antigenic Peptides

The antigenic proteins or tissues for a number of deleterious immuneresponses are known. For example, in experimentally induced autoimmunediseases, antigens involved in pathogenesis have been characterized inarthritis in rat and mouse, native type-II collagen is identified incollagen-induced arthritis, and mycobacterial heat shock protein inadjuvant arthritis; thyroglobulin has been identified in experimentalallergic thyroiditis (EAT) in mouse (Maron et al. (1988), J. Exp. Med.,vol. 152:1115-1120); acetyl choline receptor (ACHR) in experimentalallergic myasthenia gravis (EAMG) (Lindstrom et al. (1988), Adv.Immunol., vol. 42:233-284); and myelin basic protein (MBP) andproteolipid protein (PLP) in experimental allergic encephalomyelitis(EAE) in mouse and rat. In addition, for example, target antigens havebeen identified in humans: type-II collagen in human rheumatoidarthritis (Holoshitz et al. (1986), Lancet ii:305-309); and acetylcholine receptor in myasthenia gravis (Lindstrom et al. (1988), supra)all of the above are incorporated herein by reference.

Antigens are presented on MHC molecules on the surface ofantigen-presenting cells subsequent to the hydrolysis of antigenicproteins into smaller peptide units. The location of these smallersegments within an antigenic protein can be determined empirically. See,e.g., U.S. Pat. No. 5,734,023. These segments are thought to be about 8to about 20 residues in length, and contain both a region recognized bythe MHC molecule and an epitope recognized by a TCR on the T cell. Oncedetermined, the relevant antigenic peptides can readily by synthesized,preferably using standard automated methods for peptide synthesis. Inthe alternative, they can be made recombinantly using isolated orsynthetic DNA sequences.

In preferred embodiments, the aAPC comprises antigens wherein theantigens are presented by MHC components for contact with andrecognition by a T cell receptor. Such antigens may be selected from thegroup consisting of a peptide, a peptide derived from the recipient forgraft versus host diseases, a cancer cell-derived peptide, a peptidederived from an allergen, a donor-derived peptide, a pathogen-derivedmolecule, a peptide derived by epitope mapping, a self-derived molecule,a self-derived molecule that has sequence identity with saidpathogen-derived antigen, said sequence identity having a range selectedfrom the group consisting of between 5 and 100%, 15 and 100%, 35 and100%, and 50 and 100%.

Examples of some antigens noted above include the peptideQKRAAYDQYGHAAFE (Seq. Id. No. 10) which is derived from E. coli dnaJp1heat shock protein. A human self-derived peptide is QKRAAVDTYCRHNYG(Seq. Id. No. 11) derived from the HLA. This peptide also has sequenceidentity with the pathogen Sequence Id. No. 10. Peptides derived fromepitope mapping include human peptides from the HA I matrix GILGFVFTL(Seq. Id. No. 12), VKLGEFYNQ (Seq. Id. No. 13) which is a HA Inucleoprotein, and PKYVKQNTLKLAT (Seq. Id. No. 14) derived from the HAII locus.

Formation of the MHC Molecule:Antigenic Peptide Complexes

Complexes between antigen-presenting molecules (e.g., MHC molecules) andantigenic peptides can be formed using any suitable technique. Theantigenic peptides can be associated non-covalently with the antigenbinding sites of the MHC molecules by, for example, mixing the twocomponents. They can also be covalently bound using any suitable linkagechemistry. In many embodiments, the antigenic peptides are mixed withthe desired MHC molecules.

Making aAPCs

PCs may be made, for example, by:

-   -   (a) obtaining an MHC:antigen complex of interest;    -   (b) combining said MHC:antigen complex with an artificial lipid        membrane to form a membrane-associated MHC:antigen complex,        (i.e., a liposome:MHC:antigen complex); and    -   (c) combining said liposome:MHC:antigen complex resulting from        step (b) with any of the following: accessory molecule, a        co-stimulatory molecule, an adhesion molecule, a modulation        molecule, and an irrelevant molecule to form an artificial APC        comprising a liposome:MHC:antigen:functional molecule:complex.        Steps (b) and (c) may be performed simultaneously. In one        embodiment of this method, step (c) is optional in whole or in        part with respect to any of co-stimulatory, adhesion,        modulation, irrelevant molecules or GPI proteins. In another        embodiment, any of the molecules of interest (MRC, accessory,        co-stimulatory, adhesion, modulation, irrelevant molecules) can        be bound to the β subunit of cholera toxin and GM-1 can be        included in the APC lipid matrix to provide a means for proper        orientation of the molecules of interest such that their active        centers are oriented to facilitate interaction with T cells and        other components external to the APC.

By “membrane-associated” is meant the non-covalent attraction betweenthe lipid molecules of a liposome and MHCs, antigens, accessorymolecules, co-stimulatory molecules, adhesion molecules, modulationmolecules, irrelevant molecules, GM-1 ganglioside molecules, and choleratoxin β subunit.

In another preferred embodiment, the artificial APCs may be made by:

-   -   (a) obtaining a spheroid solid support of interest having        affinity for non-polar regions of a phospholipid; and    -   (b) combining MHC:antigen complexes, accessory molecules such as        ICAM-1, and functional molecules (i.e., other accessory        molecules, co-stimulatory molecules, modulation molecules,        irrelevant molecules and adhesion molecules) with the        phospholipid and solid support to form a solid support        associated:membrane-bound:MHC:antigen:accessory        molecule:functional molecule complexes (i.e., solid        support:phospholipid:MHC:antigen:accessory molecule:functional        molecule complex).

In this embodiment, the solid support is preferably a glass bead ormagnetic bead. It is also preferred that the phospholipid bephosphotidylcholine. In one embodiment of this aspect, the functionalmolecules are individually optional. In another preferred embodiment,the molecules may be properly oriented by inclusion of a molecule boundto cholera toxin and GM-1 in the APC membrane matrix. In anotherembodiment the complex may include an irrelevant molecule for carrying alabel. In still other embodiments, the antigen may have a label. Instill other embodiments, a label may be noncovalently associated withthe lipid layer.

In yet more embodiments of this solid-support APC construct, the solidsupport is a glass or magnetic bead having a diameter of about betweenabout 25 to about 300 um. In still another embodiment, the solid-supportAPC construct has only lipids, cholesterol and a molecule havingaffinity for binding to an irrelevant molecule that is located onanother APC (either solid-support based or non-solid support bases).This construct allows for such molecule (which is also an “irrelevant”molecule) to float freely in the lipid layer for proper migration to aidbinding of APCs that have bound to T cells.

Artificial APC Uses and Methods

In another aspect, the present invention is directed to a method ofisolating T cells specific for an antigen of interest comprising:

-   -   (a) obtaining a biological sample containing T cells which are        specific for an antigen of interest;    -   (b) preparing an artificial APC as described herein comprising        an MHC:antigen component, wherein the antigen in said component        is said antigen of interest;    -   (c) contacting the biological sample obtained in step (a) with        the artificial APC obtained in step (b) to form an artificial        APC:T cell complex;    -   (d) removing said complex formed in step (c) from said        biological sample; and    -   (e) separating T cells specific for said antigen of interest        from said complex.

Any suitable biological sample which contains T cells specific for theantigen of interest may be used in the method. Suitable biologicalsamples containing T cells specific for an antigen of interest includefluid biological samples, such as blood, plasma and cerebrospinal fluid,and solid biological samples, such as tissue, for example, histologicalsamples. In one embodiment of the above example, the artificial APC maybe complexed to a solid support in addition to the T cell. Thecomplexing of the APC to the solid support provides a means to anchorthe APC so that it and any T cell binding to it can be preferentiallycaptured and isolated from extraneous matter. In such case, the solidsupport may be a glass or magnetic bead that is coated with a lipid monolayer that is bound to the bead by, for example, a linker. The solidsupport may additionally have noncovalently bound accessory moleculesassociated with the lipid layer such as binding molecules that recognizeand bind to irrelevant molecules associated with the artificial APC. Inanother embodiment, the binding molecules may be covalently bound to thesolid support by a linker. Additionally, the lipid layer may furtherinclude GM-1 for binding to a molecule of interest that is connected tocholera toxin subunit for orienting said molecule of interest.

By “T cells specific for an antigen of interest” is meant the T cellsexpressing receptors for a relevant target of an immune response. Asnoted above, the specificity of the T cell receptor (TCR) determineswhich antigens bind to the TCR with sufficient affinity to activate aparticular T cell. Optionally, the above outlined method of isolating Tcells specific for a particular antigen of interest may includedetermining the quantity of such T cells that bind to the artificialAPC, or may include characterizing the functional phenotype of such Tcells. Such method may also include the use of a solid supportcontaining a molecule which is able to recognize and bind to theirrelevant molecule located in the lipid layer of the APC. Such solidsupport may further be confirmed to an accessible chamber of a columndevice.

By “MHC:antigen component” is meant MHC molecules that have affinity forantigens of interest which antigens are associated with such MHC andtogether form a complex of molecules that are inserted into or areassociated with the lipid membrane. Artificial lipid membranes such asliposomes may be prepared in a fashion similar to methods known in theart, (e.g., Watts, et al., PNAS, vol. 81:7564-68; Buus, et al., Cell,vol. 47;1071:77). In a preferred embodiment, the liposome forms abilayer having eukaryotic cell-like properties in that non-lipidmolecules (e.g., MHC:antigen complexes, GM-1 bound to cholera-moleculeof interest) may freely migrate within the matrix of the lipid moleculesof the liposome's lipid bilayer. An example of a liposome:MHC:antigencombination is provided in FIG. 1 and such a complex may also beprepared, for example, as described in Example 1 below. Moreover, thelipid bilayer may also include accessory molecules such as cholesterolto provide elasticity in the bilayer and GM-1 to provide an anchor fororientation of cholera β subunit comprising molecules of interest.

In one embodiment, a biological sample derived from a tissue sample ofinterest in the form of a single cell suspension is contacted with anartificial APC followed by incubation of the artificial APC tissuesample mixture with antigen-specific staining compounds (i.e. forexample, fluorochrome conjugated antibodies against T cell surfacemarkers of interest e.g., CD3, CD4, or CD8). The cells may also bestained prior to incubation with the complexes. The resulting artificialAPC:T cell complex may be separated from the cell suspension by, forexample, flow cytometry, capture by a solid support in a column device,or centrifugation of such complexes bound to a solid support such asglass beads or magnetic beads.

In one embodiment of the T cell isolation method, the antigen is labeled(FIG. 2). In another embodiment, the irrelevant molecule may be labeled(FIG. 3). In another embodiment of the method, label is associatedeither covalently or non-covalently with the lipid molecules making upthe liposome (FIG. 4). Preferred labels include biotin, fluorochromessuch as FITC, radioactive labels and vancomycin. Use of such labels iswell understood in the art. In embodiments where the label is associatedwith lipids of the liposome, the label may be either enclosed within theliposome or incorporated within the lipids of the outer membrane of theliposome. Preferably, the label is a fluorochrome, for example FITC.Such labeled liposomes may be made by mixing FITC with lipids duringliposome formation, or may be obtained from commercial sources (e.g.,polar lipids).

In another embodiment, the present invention provides an alternatemethod of isolating T cells specific for an antigen of interest. Thisalternate method comprises:

-   -   (a) contacting an artificial APC comprising a        MHC:antigen:accessory molecule component of interest with a        solid support to form a solid support:artificial APC (The        liposome of the APC contains a binding molecule i.e., an        “irrelevant” binding molecule. The capture molecule that binds        to the irrelevant molecule may be bound to said solid support        via a linker or may be associated with a phospholipid layer on        the solid support). In this embodiment, the antigen binding        region of said MHC:antigen component is available for binding to        a T cell receptor without steric hindrance because the        MHC:antigen component is free to move within the liposome        membrane of the APC while the irrelevant binding protein allows        the APC to be anchored to the solid support;    -   (b) contacting said solid support:artificial APC with a        biological sample containing T cells specific for an antigen of        interest to form a solid support:artificial APC:T cell complex;    -   (c) removing said solid support:artificial APC:T cell complex        from said biological sample; and    -   (d) separating the T cells specific for said antigen of interest        from said complex.

In this embodiment, the T cells are separated by either the physicalremoval of the T cell bound solid support from the biological sample, orseparation may occur by retention of the bound T cells in a solidsupport containing compartment of a column device.

In another embodiment, the present invention is directed to otheralternate methods of isolating T cells specific for an antigen ofinterest. One such method comprises:

-   -   (a) obtaining a solid support of interest having affinity for        non-polar regions of a phospholipid;    -   (b) combining the solid support and MHC:antigen complexes,        accessory molecules such as LFA-1, and functional molecules        (i.e., other accessory molecules, co-stimulatory molecules,        modulation molecules, adhesion molecules, and irrelevant        molecules) with the phospholipid support to form a solid support        associated:membrane-bound:MHC:antigen:accessory        molecule:functional molecule complexes (i.e., solid        support:phospholipid:MHC:antigen:accessory molecule:functional        molecule complex;    -   (c) contacting said solid        support:phospholipid:MHC:antigen:accessory molecule:functional        molecule complex formed in (b) with a biological sample        containing T cells specific for an antigen of interest to form a        solid support:phospholipid:MHC:antigen:accessory        molecule:functional molecule:T cell complex;    -   (d) removing said complex formed in (c) from said biological        sample; and    -   (e) separating the T cells specific for said antigen of interest        from said complex.

In this embodiment, the solid support is preferably a glass bead ormagnetic bead of about 25 to about 300 um in diameter. Additionally, theMHC, accessory, and functional molecules are not covalently bounddirectly to the solid support but are noncovalently associated with thelipid layer having the capacity to migrate on the surface of the solidsupport. Moreover, in a preferred embodiment of this example, themolecule of interest (e.g., MHC, accessory and functional molecules) areconnected to cholera toxin β subunit. Further, GM-1 protein isincorporated into the lipid layer matrix providing a means by which thecholera toxin portion can be bound and the molecule of interest properlyoriented in the lipid layer.

In still another aspect, a spheroid solid support may comprise lipidmonolayer coating the solid support with only a irrelevant moleculehaving affinity for bonding another irrelevant molecule located on anAPC that either does not have a solid support interior or does have asolid support interior. This aspect adds versitility to the captureprocess for isolation of APCs that are bound to antigen-specific Tcells.

In a further preferred aspect of the invention, kits are provided forthe isolation of T cells specific for an antigen of interest comprisingany of the following: solid supports, phospholipids, antigen-specificartificial APCs, TCR-specific artificial APCs, solid supports containinglipid associated capture molecules for capturing irrelevant molecules,solid supports containing capture molecules bound to the solid supportbuffers, media, labels and column devices.

In another embodiment, the invention contemplates a method ofcharacterizing the functional state of antigen-specific T cellscomprising:

-   -   (a) isolating T cells;    -   (b) extracting mRNA from said isolated T cells;    -   (c) obtaining cDNA corresponding to said extracted mRNA;    -   (d) evaluating the mRNA encoding proteins that govern function        and phenotype of the antigen-specific T cells wherein the        evaluation is carried out by a method selected from the group        consisting of (1) mRNA translation of the proteins and testing        such proteins using antibodies against the proteins, and (2)        rtPCR of the mRNA using primers specific for the proteins.

In this method, the evaluation of the mRNA encoding proteins that governfunction and phenotype of the antigen-specific T cell may be used todetermine efficacy of an immunomodulation treatment regimen such as theadministering of a vaccine. The immunomodulation treatment can compriseinducing tolerance in autoimmunity, reducing allergic response, inducingan immune response against cancer cells. Additionally, the proteins thatgovern function and phenotype of the antigen-specific T cells includecytokines, chemokines, chemokine receptors, and cytokine receptors.

The genes encoding antigens may also be identified. In another aspect,the invention contemplates a method for identifying a gene which isexpressed by a T cell specific for an antigen of interest comprising:

-   -   (a) obtaining a biological sample containing T cells which are        specific for an antigen of interest;    -   (b) labeling with a first label at least the intracellular gene        product of interest produced by T cells in said biological        sample;    -   (c) preparing a liposome:MHC:antigen complex, wherein the        antigen in said liposome:MHC:antigen complex is said antigen of        interest;    -   (d) contacting the biological sample obtained in step (a), as        labeled in accordance with step (b), with the liposome:MHC:        antigen complex obtained in step (c) to form a        liposome:MHC:antigen:T cell complex;    -   (e) labeling with a second label the liposome:MHC:antigen:T cell        complex obtained in step (d); and    -   (f) discriminating, according to antigen specificity, cells        producing the intracellular gene product of interest, which        cells have both the first label and the second label.

In such method, the first and said second label may be selected from thegroup consisting of biotin, a flurochrome, FITC, and a radioactivelabel; provided that the first and second labels are not the same.

In yet another preferred embodiment, methods are provided for modulatingT cell responses. In this embodiment, T cells are isolated from asubject which are specific for an antigen of interest followed bycombining said isolated T cells with an artificial APC having functionalmolecules specific for modulating a T cell. In a preferred embodiment,the T cells specific for an antigen of interest are isolated using anyof the T cell isolation methods described herein. In one preferredembodiment, modulation to activate T cells generally is caused bycontacting said T cells with an artificial APC that expresses theco-stimulatory molecule B7 or anti-CD28, as well as MHC:antigen.

By “modulating T cell response” is meant the intentional intervention inthe functional characteristics of antigen-specific T cells, including,but not limited to, functional pattern of cytokines produced, change inphenotype of T cells, and modulation in the expression of activationmarkers, cytokines and their receptors and chemokines and theirreceptors. Such modulations can be carried out for numerous purposes asdiscussed in the numerous examples above.

The modulation of T cell response may further comprise changing in wholeor in part the functional pattern of cytokine production by the isolatedT cells specific for a given antigen from a Th1 response to a Th2response. In a preferred embodiment, modification of a T cell responsefor the purpose of increasing its Th2 response and/or decreasing its Th1response includes the expression by the artificial antigen presentingcell used in such method of a co-stimulatory molecule, such as B7-2.

In another embodiment, the modulation of T cell response may comprisechanging in whole or in part the functional pattern of cytokineproduction by said isolated T cells from a Th2 response to a Th1response. Preferably, modulation of a T cell response for the purpose ofincreasing its Th1 response and/or decreasing its Th2 response includesthe expression by the artificial APC used in such method of aco-stimulatory molecule such as B7-1.

The two subsets of CD4 T cells represented by Th1 and Th2 cytokinemarkers, have very different functions from one another. These two CD4 Tcell subsets can also regulate each other. Once one subset becomesdominant, it is often difficult to shift the response of the T cell(i.e. expression of the cytokine) to the other type. One reason for thisis that expressed cytokines of one type of CD4 T cell will inhibit theactivation of the T cell to expression of another cytokine. For example,IL-10, a product of Th2 cells, can help to inhibit the development of aTh1 response. Therefore, in one embodiment, IL-10 is incorporated intoartificial APCs and the resulting APC may be used to inhibit developmentof Th1 response.

In another example, interferon-γ (INF-γ), a product of Th1 cells, canhelp to prevent the activation of a Th2 response. If a particular CD4 Tcell type is activated preferentially in a response so that the cytokine(Th1 or 2) is highly expressed, such high expression can suppress thedevelopment of the other subset. The overall effect of T cellpopulations expressing one or the other subset is that various tissuesbecome either suppressive (Th2) or inflammatory (Th1). Thus, INF-γ canbe incorporated into artificial APCs and the resulting APC used tomodulate cell populations to become either suppressive or inflammatory.

The ability to manipulate T cell response (as exemplified by the Th1/Th2subsets) provides a novel method by which treatment can be provided fornumerous disease states. For example, a response to an allergen can bemanipulated so as to shift the antibody response away from anIgE-dominated response. Such a shift will prevent the allergen fromactivating IgE-mediated effector pathways. For example, a technique thathas been used for many years to generate a desensitization response iscarried out by contacting patients with escalating doses of the allergen(such as by feeding, nasal delivery, or by injection). This immunizationschedule appears to gradually divert an IgE-dominated response, whichdiversion is driven by Th2 cells, to one driven by Th1 cells, with theconsequent down-regulation of IgE production. Thus, the activation of aTh2 response in such a manner may be of use in the treatment of allergy.

Similarly, other conditions that are associated with a Th2 T cellresponse, as for example, responses in which the functional phenotype ofcytokine secretion is tolerogenic to viral infections and some types ofcancer, may also benefit from a modification of the Th2 T cell responseso that Th2 is reduced and/or a Th1 T cell response is increased.

Autoimmune conditions, in which the functional phenotype of cytokinesecretion is pro-inflammatory (as is the case with Th1), will benefitfrom a modification of the Th1 T cell response so that it is reducedand/or a Th2 T cell response is increased. As noted above, cytokines arecell-derived soluble mediators associated with immune responses that mayact both within a microenvironment and/or systemically. Immune cellssecrete specific cytokine profiles, each having markedly differenteffects. Th2-type cells secrete low levels of TGF-β, no IFN-γ and highlevels of IL-4 and IL-10, the presence of which in turn produce animmunosuppressive or tolerogenic immune environment. Th1-type cells onthe other hand secrete very low TGF-β, high IFN γ and no IL-4 or IL-10.High expression of INF-γ is associated with cell mediatedproinflammatory immune environments. As discussed herein, the artificialAPC of the current invention can be used to modulate T cell responses byaffecting these and other cytokines. Moreover, such cytokines as well asother soluble factors to which a T cell responds may be used inconjunction with a column device as described below.

In yet another embodiment, the regulation of T cell responses maycomprise inducing anergy. Specifically, T cell responses may be modifiedfor the purpose of inducing anergy through the Fas/Fas Ligand pathway.

In yet another aspect, the present invention provides methods oftreating a condition in a subject who would be benefited by modulatingthe functional patterns of cytokine production by certain of suchsubject's antigen-specific T cells to increase Th2 response and/ordecrease Th1 response, comprising:

-   -   (a) isolating T cells capable of triggering a Th1 response from        a subject;    -   (b) combining said isolated T cells with artificial APCs which        express MHC capable of binding an antigen recognized by said T        cells wherein said artificial APC also expresses the        co-stimulatory molecule B7-2;    -   (c) separating T cells that have bound to said artificial APCs        in step (b); and    -   (d) administering said T cells isolated in step (c) to said        subject.

T cells that have been in contact with an artificial APC as describedabove will be stimulated to shift their Th1 response to a Th2 response.Conditions which would be benefited by modulating the functional patternof cytokine production to increase Th2 response and/or decrease Th1response include autoimmune diseases such as, for example, type 1diabetes mellitus, multiple sclerosis, rheumatoid arthritis,dermatomiosytis, juvenile rheumatoid arthritis and uveitis.

In yet another aspect, the present invention provides methods oftreating a condition in a subject that would be benefited by alteringthe functional pattern of cytokine production by certainantigen-specific T cells to increase Th1 response and/or decrease Theresponse comprising:

-   -   (a) isolating T cells that are specific for an antigen capable        of triggering a Th2 response from a subject;    -   (b) combining said isolated T cells with an artificial APC which        expresses an MHC capable of binding an antigen that is        recognized by said T cells so that the functional pattern of        said T cells may be modulated, wherein said artificial APC also        expresses the co-stimulatory molecule B7-1;    -   (c) separating the T cells modulated in step (b); and    -   (d) administering said T cells isolated in step (c) to said        subject.

Conditions which would be benefited by modulating the functional patternof cytokine production to increase Th1 response and/or decrease Th2response include for example, allergy, allergy to dust, animal skinbypass products, vegetables, fruits, pollen, chemicals, and someinfections (e.g., viral, fugal, protozan and bacterial).

As noted above, the present invention provides methods for isolatingantigen-specific T cells. It is desirable to isolate antigen-specific Tcells in a variety of contexts. For example, in adoptive immunotherapy,lymphocytes are removed from a patient, expanded ex vivo, and thenreinfused back into the patient to augment the patient's immuneresponse. See Rosenberg et al., N. Engl. J. Med. 313:1485-1492 (1985);U.S. Pat. No. 4,690,915. This approach has been effective in thetreatment of various cancers (see, e.g., Rosenberg et al., N. Engl. J.Med. 319:1676-1680 (1988). Isolation and expansion of T cells specificfor a particular antigen will increase the specificity and effectivenessof adoptive immunotherapeutic approaches. Such expansion may bebenefited by obtaining a population of monoclonal T cells. Thus, theinvention contemplates a method of obtaining a monoclonal population ofT cells specific for an antigen of interest comprising:

-   -   (a) isolating T cells specific for an antigen of interest; and    -   (b) culturing the isolated T cells in an individual well with        the antigen of interest and an artificial APC.

Isolated T cells specific for a particular antigen may also be used as adiagnostic to screen for the presence of, or amount of a particularantigen and thus detect the presence, absence, or status of an immuneresponse. Early detection of an immune response will facilitateselection of a particular treatment regimen in a variety of pathologicalconditions such as autoimmune diseases, allergies, allograft rejection,and infectious diseases. In a diagnosis of this type, the isolated Tcells are used both as a means of detection and as reporters. The Tcells proliferate when contacted with the antigen for which they arespecific. This proliferation is easily detected as an increase in cellnumber or as an increase in growth rate measured, for example, by therate of uptake of a label (e.g., observation of the uptake of tritiatedthymidine or bromodeoxyuricyl (BrdU)). Thus, the presence or absence oftarget antigen can be detected by exposing the isolated T cells to atissue sample (e.g., peripheral blood) and monitoring theirproliferation rate. In the current invention, a preferred embodimentincludes the observation of bound T cells using a FACS which allows theavoidance of time consuming proliferation experiments.

Isolation of antigen-specific T cells also provides a homogenous sourceof T cell receptors. A homogenous source of T cell receptors is an aidto the elucidation of structure-function relationships of particularreceptors. A homogenous source of T cell receptors also facilitates thedevelopment of solubilized T cell receptors that are of use in a numberof therapeutic applications (See, e.g., U.S. Pat. No. 5,283,058). In thecurrent invention, solubilized receptors may be used in conjunction withan immunomodulation column device.

In another preferred embodiment, the invention provides for a method ofidentifying epitopes expressed on the MHC that are of import toacceptance or rejection of grafts in transplantation therapy. In thisembodiment, a donor's MHC is examined by computer modeling to identifypeptide moieties likely to be recognized by a recipient's T cells. Therecipient's T cells are tested by FACS analysis for binding against thepeptides. Upon positively identifying reactive peptides, such peptidesmay then be used to deplete the recipient's graft rejecting T cellsthereby modulating the recipient's immune response in thetransplantation regimen. This modulation is carried out within acomprehensive treatment that includes further modulating the recipient'ssensitivity to the graft epitopes by exposing the recipient to thedonor's epitopes by feeding, nasal delivery, or injection of increasingconcentrations of the peptides.

In another aspect, the invention provides for a method to identifyantigenic motifs of pathogens that are recognized by the MHC. In thisembodiment the identification of such motifs allows the development ofanti-pathogen vaccines comprising either pools of such motifs in theform of peptides that are recognized by T cells in the general humanpopulation i.e., the pathogen's antigenic moieties responsible forgenerating immune responses are identified so that for any disease, avaccine comprising such antigens may be produced. Additionally, avaccine may be produced against such antigenic moieties comprising apopulation of a patient's pathogen-specific T cells that have beenexpanded ex vivo.

In yet another aspect of the invention, a column device is providedcomprising a multiplicity of compartments that are arranged in series.As shown in FIG. 8, one embodiment of such device has compartments A, B,and C. Each compartment is contiguous with the adjacent compartment by avalve 1. The valve between each compartment may be opened or closed.Compartment A also has entrance ports 2 and exit ports 3 for receiving aflowable medium. Any of the compartments may contain solid supports asseen in FIGS. 6, and 7 for binding either a molecule that can bind to anirrelevant molecule for immobilizing an artificial APC, or for bindingdirectly MHC:antigen:functional molecule complexes. The entrance port 4of compartment A is attached to an external device 5 (e.g., a devicewhere a patient's T cells are separated from whole blood andconcentrated) prior to export through a sterile tube 6 to entrance port4. Once the patient's enriched T cell population is transported intocompartment A via entrance port 4, the T cells containing TCR specificfor the MHC:antigen complexes bind to MHC:antigen complexes that arethemselves attached either directly to the solid supports or areassociated with an artificial APC which is bound to a solid support viaan irrelevant molecule. In the configuration demonstrated in FIG. 8,artificial APCs 14 are attached to solid supports in compartment Aawaiting interaction with incoming cell through interance port 4.Compartment A thus provides a chamber wherein the APCs and cells caninteract so that antigen-specific T cells can be isolated whilenon-binding T cells are allowed to wash out of the compartment A viaexit port 3 a. After the non-binding cells are removed (and eitherdiscarded or returned to the patient's circulatory system, or addressedto a device for monitoring the cells such as a FACS), the boundantigen-specific T cells may be released from the solid supports in Aand allowed to channel or flow through valve 1 located betweencompartment A and B and into compartment B. The artificial APC-cellbound complexes or just the antigen-specific T cells alone may bereleased from solid support of compartment A by adjusting thetemperature of the medium in the compartment A. Once theantigen-specific T cells are in compartment B, they may again becaptured as shown in FIG. 8, or simply be treated without recapture inany number of ways to induce cell modulation. For example, artificialAPCs containing antigen-specific MHC:antigen:functional moleculecomplexes may be infused into compartment B via entrance port 7. Withthe infusion of the antigen-specific APCs into compartment B, theantigen-specific T cells may then bind and be induced to modulate theirrespective response or be acted upon otherwise as desired. FIG. 8 showsT cells 13 that have been released from compartment A and are shownbound to APCs that are in turn bound to solid supports. As describedabove, and hereinafter, numerous types of modulation of the T cells mayoccur. For example, the T cells maybe modulated to (1) increase a Th1and/or decrease a Th2 response, (2) increase a Th2 and/or decrease andTh1 response, (3) be generally induced to proliferate regardless of Th1or Th2 response, (4) enter a state of anergy, (5) become apoptotic, (6)shed certain adhesion molecules known to those skilled in the art to beinvolved in the entry of cells into specific diseased areas, (7)upregulate/downregulate chemokine or cytokine receptors associated witha Th1-type response, (8) upregulate/downregulate chemokine or cytokinereceptors associated with a Th2-type response. The T cells may also becaptured on such APCs for the purpose of (1) depletingallo-antigen-specific T cells from a donor's T cell population intransplantation oriented therapy, such as bone marrow transplant therapyor (2) from a patient's T cell population such as to facilitate a graftof allogenic solid organs, or (3) used in identifying TCRs that bind topathogen-recognizing MHCs or self-derived pathogen-mimic antigenicmotifs. Moreover, soluble molecules may be added to the device forinducing such modulation including cytokines, adjuvants, and hormones.

Once isolation or other modulation has been performed in compartment B,the T cell:artificial APC complex may be addressed to compartment C viathe valve 1 between compartments B and C. The entrance port 7 ofcompartment B may be additionally connected to external units forsupplying buffers and the like that may be necessary for carrying outmanipulations of the T cells in compartment B. The exit port 8 ofcompartment B may also be used to transport modulated T cell:artificialAPC complexes to sampling devices such as FACS or to other devices forsuch things as cell proliferation and the like.

Compartment C may also contain solid supports and the like for bindingartificial APC:T cell complexes and further treatment. In compartment C,the modulated and isolated T cells may be eluted from the APCs (via theadjustment of the medium's temperature) and addressed to other devicesthrough ports 9 and 10, or addressed out of the column through exit port11 to, for example, the patient, a FACS, a culture device, or to anotherlocation for further manipulation.

Formulation and Administration

For in vivo application, aAPCs of the invention are administered by anysuitable route, preferably by injection. For systemic administration,intravenous injection is preferred. Suitable formulations are found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985). A variety of pharmaceuticalcompositions comprising aAPCs of the present invention andpharmaceutically effective carriers can be prepared. The pharmaceuticalcompositions are suitable in a variety of drug delivery systems.

In preparing the pharmaceutical compositions of the present invention,it is frequently desirable to modify the aAPCs to alter theirpharmacokinetics and biodistribution. For a general discussion ofpharmacokinetics, see, Remington's pharmaceutical sciences, supra. Anumber of methods for altering pharmacokinetics and biodistribution areknown. For instance, methods suitable for increasing serum half-life ofaAPCs include treatment to remove carbohydrates which are involved inthe elimination of substances from the bloodstream. Also, conjugation tosoluble macromolecules, such as proteins, polysaccharides, or syntheticpolymers, such as polyethylene glycol, is also effective. Preferredmacromolecules include those that target aAPCs complexed therewith to aspecific tissue (e.g., a lymphoid organ) or cell type. A number of suchtargeting molecules are known in the art, and they include proteins andcarbohydrates.

Liposomes can be prepared according to any suitable method known in theart. Following liposome preparation, the liposomes may be sized toachieve a desired size range and relatively narrow distribution ofliposome sizes. One preferred size range is about 0.2-0.4 um, whichallows the liposome suspension to be sterilized by filtration through aconventional filter, typically a 0.22 micron filter.

Several techniques are available for sizing liposome to a desired size.These include sonicating a liposome suspension either by bath or probesonication to produce a progressive size reduction. Homogenization isanother method, and it relies on shearing energy to fragment largeliposomes into smaller ones. In a typical homogenization procedure,multilamellar vesicles are recirculated through a standard emulsionhomogenizer until selected liposome sizes, typically between about 0.1and about 0.5 microns, are observed. In such methods, particle sizedistribution can be monitored, for example, by conventional laser-beamparticle size discrimination.

Extrusion of liposomes through a small-pore polycarbonate membrane or anasymmetric ceramic membrane is also an effective method for reducingliposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in liposome size.

Even under the most efficient encapsulation methods, the initial sizedliposome suspension may contain various components that have not beenincorporated into a liposome. If desired, any suitable available forremoving non-entrapped compounds from a liposome suspension may beemployed. In one such method, liposomes are pelleted by high-speedcentrifugation, leaving free compounds and other contaminants in thesupernatant. Another method involves concentrating a liposome suspensionby ultrafiltration, then resuspending the concentrated liposomes in areplacement medium. Alternatively, gel filtration can be used toseparate large liposome particles from solute molecules.

After arriving at a desired aAPC composition based on liposomes, theliposome suspension may be brought to a desired concentration foradministration. This may involve resuspending the liposomes in asuitable volume of injection medium, where the liposomes have beenconcentrated, for example by centrifugation or ultrafiltration, orconcentrating the suspension, where the drug removal step has increasedtotal suspension volume. The suspension may then be sterilized byfiltration. The composition may then be administered, for example,parenterally or locally in a dose which varies according to, e.g., themanner of administration, the particular disease being treated, etc.

For pharmaceutical compositions which comprise aAPCs of the presentinvention, the dose will vary according to, e.g., the particular aAPCcomposition complex, the manner of administration, the particulardisease being treated and its severity, the overall health and conditionof the patient, and the judgment of the prescribing physician.

The pharmaceutical compositions are intended for parenteral, topical,oral, or local administration, such as by aerosol or transdermally, forprophylactic and/or therapeutic treatment. The pharmaceuticalcompositions can be administered in a variety of unit dosage formsdepending upon the method of administration. For example, unit dosageforms suitable for oral administration include powder, tablets, pills,and capsules.

Preferably, the pharmaceutical compositions are administeredintravenously. Thus, this invention provides compositions forintravenous administration that comprise a solution of aAPCs dissolvedor suspended in a pharmaceutically acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.4% saline, and the like. For instance, phosphatebuffered saline (PBS) is particularly suitable for administration ofaAPCs of the present invention. These compositions may be sterilized byconventional, well-known sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

The concentration of the aAPCs in the composition can vary widely, i.e.,from less than about 0.05%, usually at or at least about 1% to as muchas 10 to 50% or more by weight and will be selected primarily by fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected. Preferred concentrations for intravenousadministration are about 0.02% to about 0.1% or more in PBS.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient.

For aerosol administration, the aAPCs are preferably supplied in finelydivided form along with a surfactant and propellant. The surfactantmust, of course, be nontoxic, and preferably soluble in the propellant.Representative of such agents are the esters or partial esters of fattyacids containing from 6 to about 22 carbon atoms, such as caproic,octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric andoleic acids with an aliphatic polyhydric alcohol or its cyclic anhydridesuch as, for example, ethylene glycol, glycerol, erythritol, arabitol,mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, andthe polyoxyethylene and polyoxypropylene derivatives of these esters.Mixed esters, such as mixed or natural glycerides may also be employed.The surfactant may constitute 0.1%-20% by weight of the composition,preferably 0.25-5%. The balance of the composition is ordinarilypropellant. Liquefied propellants are typically gases at ambientconditions, and are condensed under pressure. Among suitable liquefiedpropellants are the lower alkanes containing up to five carbons, such asbutane and propane. Mixtures of the above may also be employed. Inproducing the aerosol, a container equipped with a suitable valve isfilled with the appropriate propellant, containing the finely dividedcompounds and surfactant. The ingredients are thus maintained at anelevated pressure until released by action of the valve.

The compositions containing aAPCs can be administered for therapeutic,prophylactic, or diagnostic applications. In therapeutic applications,compositions are administered to a patient already suffering from adisease, as described above, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Anamount adequate to accomplish this is defined as “therapeuticallyeffective dose.” Amounts effective for this use will depend on theseverity of the disease and the weight and general state of the patient.As discussed above, this will typically be between about 0.5 mg/kg andabout 25 mg/kg, preferably about 3 to about 15 mg/kg.

In prophylactic applications, compositions containing aAPCs of theinvention are administered to a patient susceptible to or otherwise atrisk of a particular disease. Such an amount is defined to be a“prophylactically effective dose.” In this use, the precise amountsagain depend on the patient's state of health and weight. The doses willgenerally be in the ranges set forth above.

Kits can also be supplied for in vivo uses. Often, the subjectcomposition of the present invention will be provided, usually in alyophilized form, in a container. The aAPC-containing compositions areincluded in the kits with buffers, such as Tris, phosphate, carbonate,etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or thelike, and a set of instructions for use. Generally, these materials willbe present in less than about 5% wt. based on the amount of complex andusually present in total amount of at least about 0.001% wt. based againon the protein concentration. Frequently, it will be desirable toinclude an inert extender or excipient to dilute the active ingredients,where the excipient may be present in from about 1 to 99% wt. of thetotal composition. A suitable buffer for reconstituting the drycomposition is also provided in the kit.

Specific embodiments of the present invention are exemplified in thefollowing Examples. These Examples are not to be interpreted as limitingthe scope of the invention in any way, the scope being disclosed in theentire specification and claims.

EXAMPLE 1 Liposome Assay for Detection of Antigen-Specific T Cells

In this example, experiments are described which demonstrate thecapacity of T cells to bind to liposomes containing cholesterol havingMHC:antigen complexes inserted into the liposome membrane. The capacityof T cell binding was quantified by flow cytometry analysis (FACS).Negative controls for the binding include the use of a control T cellline (i.e., non-reactive) having specificity for an irrelevant peptide,incorrect MHC restriction, peptide and antibody inhibitions, limitingdilutions, and the use of MHC without peptide.

The ability of the method to provide for discrimination betweenantigen-specific T cells was facilitated by use of two T cell hybridomasspecific for the same peptide. These hybridomas were OVA³²³⁻³³⁶ (whichcorrespond to residues 323-326 of ovalbumin) (obtained from ResearchGenetics, Huntsville Ala.) which were restricted by two different MHCs,I-A^(S) and I-A^(d). Specifically, the designations for the restrictionswere I-A^(s) restricted OVA³²³⁻³³⁶ specific T cell hybridoma, AG111.207,and the Iα52-restricted OVA³²¹⁻³³⁶ specific T cell hybridoma 8D051.15. Apeptide containing 2 identities and one conservative substitution, Hi15(Research Genetics), which corresponds to residues 15-31 of H.influenzae isoleucyl tRNA transferase, was used as a negative control.

Materials and Methods Preparation of Liposome:MHC:Antigen Complexes

Liposomes were prepared similarly to that described by Brian et al.,PNAS, 81:6159-63. Briefly, cholesterol (Ch) and L-α-phosphotidylcholine(PC) (Sigma) were mixed at a molar ratio of 2:7 of Ch and PCrespectively. The mixture was placed under an argon stream for 30minutes to evaporate chloroform used in the preparation, and resuspendedin 140 mM NaCl, 10 mM Tris HCl, and 0.5% deoxycholate at pH 8. Thesuspension was sonicated for three minutes or until clear.

Complexes of affinity-purified MHC molecules I-A^(s) and I-A^(d) (eachexpressed in a B cell lymphoma and purified via immunoaffinity column)were inserted into liposomes by a 72 hour 4° C. dialysis against threechanges of PBS (Slidalyzer, Pierce) at a 1:10 molar ratio of MHC toliposomes to form liposome:MHC complexes.

The OVA³²³⁻³²⁶ peptide and the control peptide, Hi15, were biotinylated,(post synthesis, Sigma), and the biotinylated peptides (b-peptides) wereincubated with the liposome:MHC complexes for 18 hours at roomtemperature at a physiologic pH to form liposome:MHC:b-peptidecomplexes.

Flow Cytometry

Viable cells were separated from debris using a ficol-hypaque gradient.(Lymphocyte M) Cells were blocked with 10% FCS in PBS for 10 minutes onice, then washed in PBS. Incubations with antibodies (used atconcentrations of 400-600 ng/ml) were performed on ice in the dark for20 minutes. The antibodies used included anti-CD3e (clone 145-2C11),anti-CD4 (clone GK 1.5), anti-CD8a (clone 53-6.7), anti-HSA (cloneM1/69), and anti-CD69 (H1.2F3) (Pharmingen, San Diego, Calif.).Liposome:MHC:b-peptide complexes were preincubated with fluorescentstreptavidin molecule (f-strep) at room temperature for ½ hour. When twopeptides were used in the same assay, the liposome:MHC:b-peptide complexwas preincubated with streptavidin molecules of differing fluorescenceprior to addition to cells. Sorted cells were either (a) cultured (usinga 3:1 ratio of irradiated BALB/c spleen cells, 20 U/ml rIL-2, 10 ug/mlHi15 or Iα52, 10% FCS, 1% Penicillin/Streptomycin/Glutamine (P/S/G), inRPMI 1640 at 37° C. and 6% CO₂), (b) processed for variable beta chain(Vb) analysis by PCR, or (c) reanalyzed by a fluorescent antibody cellsorter (FACS).

Yields ranged from 2,000 to 16,000 events. Bulk-sorted cells used forreanalysis were incubated for ½ hour on ice and spun down through 100%FCS at 325 times.g for 10 minutes to remove liposome:MHC:b-peptidecomplexes, prior to restaining with liposome:MHC and a differentb-peptide. Single-cell sorts were dispersed in 96-well culture platescontaining fresh irradiated APCs obtained from the spleen of a syngeneicBALB/c mouse. Generally 8-12 wells showed proliferation over six weeks.Cells were visualized with a Becton Dickinson FACS Star equipped withLYSIS II software.

As shown in FIG. 9, specific recognition of MHC/peptide complexes by T-Thybridomas AG111.207 (I-A^(s)/OVA³²³⁻³³⁶ specific) and 8D051.15(IA^(d)/OVA³²³⁻³³⁶ specific) were observed. Cultured AG111.207 or8D051.15 cells were analyzed by flow cytometry using anti-CD4 antibodiesand I-A^(d)/b-peptide or I-A^(s)/b-peptide combinations complexed intoliposomes and visualized by addition off-strep. The ratio of cellsspecific to either the I-A^(d)/antigen or A^(s)/antigen was constantbetween experiments. (All figures are gated on CD4+ cells unlessotherwise stated.) FIG. 9(a) shows AG111.207 cells which were stainedwith anti-CD4 antibody and differing combinations of MHC/antigenincluding an irrelevant peptide known to bind to I-A^(d) (Hi15).Cellular specificity is shown by use of a B cell hybridoma (FIG. 9-a 4).FIG. 9(b) shows 8DO51.15 cells which were analyzed with differingcombinations of MHC:antigen. Included is a control using biotinylatedI-A^(d) alone to detect the T cell hybridoma (FIG. 9-b 4).

Results

In the series of experiments for which results are shown in FIG. 9,purified I-A^(s) or I-A^(d) MHCs were inserted into liposomes and thencomplexed to the biotinylated OVA³²³⁻³³⁶ peptide. These complexes wereincubated with streptavidin-FITC, and then with a standard amount ofAG111.207 or 8D051.15 cells (final MHC concentration of 66 ug/ml). Whenanalyzed by flow cytometry, nearly 90% of the AG111.207 (FIG. 9 a 1) and87.2% of 8D051.15 (FIG. 9 b 1) cells stained positive when using thecorrect restriction and peptide.

The specificity of the entire interaction was demonstrated by lack ofstaining of AG111.207 and 8D051.15 cells when incubated with anti-CD4antibodies and complexes of the incorrect restriction for eachhybridoma, I-A^(d) and I-A^(s) respectively, and Hi15 which has twoidentities (p2, p10) and one conservative substitution (p5) withOVA³²³⁻³³⁶ (0% positive cells FIG. 9 a 2; 2.5% positive cells, FIG. 9 b2). The peptide specificity of the interaction was demonstrated whenAG111.207 and 8D051.15 cells were incubated with anti-CD4 antibody, thecorrect restriction element, but irrelevant peptide for each hybridoma,I-A^(s)/b-Hi15 or I-Ad/b-Hi15 respectively (4.6% positive cells, FIG. 9a 3; 8.7% positive cells, FIG. 9 b 3). The binding between MHC:b-peptidecomplexes and AG111.207 T cells was also concentration dependent. Only13.1% of AG111.207 cells tested positive when the I-A^(s)/OVA³²³⁻³³⁶concentration in the assay was reduced five fold to 13 ug/ml (notshown). The signal was also reduced by the addition of 300 ug/ml of thesame, non-biotinylated OVA³²³⁻³³⁶ peptide as a competitive inhibitorduring preparation of the I-A^(s)/OVA complexes (5.1% of CD4+ cellspositive, not shown). This finding suggests that biotinylation of thepeptide does not interfere with the trimolecular interactions amongpeptide, MHC and TCR. Moreover, this assay was seen to be dependent onboth TCR and MHC:peptide complexes, insofar as binding can be inhibitedby simultaneous addition of anti-TCR and anti-I-A antibodies (6.9% ofCD4+ cells positive). As shown in FIG. 9 a 4, no binding to TCR-negativecells, such as B cell hybridoma HT.01, was detected (0% of cellspositive). Using biotinylated I-A^(d) in liposomes without peptide, 6.9%of 8D051.15 cells bound the MHC alone (FIG. 9 b 4). Hence, theinteraction requires the presence of the specific peptide.

Artificial APCs Identify T Cell Hybridoma 8DO, Specific for IA^(d)/OVACombination

In another experiment we evaluated the capability of artificial APC,presenting synthetic biotynilated peptide OVA in the context of IA^(d),to visualize by FACS analysis hybridoma 8D0, which is OVA/IA^(d)specific. As shown in FIG. 15, the percentage of hybridoma cells wasvisualized by binding with cychrome-tagged artificial APC. Thisinteraction was specific, insofar as TCR binding was dependent on theavailability of the MHC/peptide complexes. The interaction wasinhibitable by addition of antibodies interfering with such interaction(% positive cells, FIG. 15 middle graph), and 8DO hybridoma cells didnot bind to the artificial APC presenting the correct peptide in thecontext of IE^(d) (% positive cells, FIG. 15 left graph). The resultindicates that highly specific and sensitive interaction occurs betweenT cells and artificial APCs. Moreover, addition of competitiveinhibitors in reducing the specific binding proves that the labeling ofthis method does not interfer with the APC/T cell interaction.

EXAMPLE 2 Identification of Antigen Specific T Cells in Mouse EmbrionicThymuses

In this example, a murine model is used to investigate the effects of anaturally processed self-peptide on the maintenance and proliferation ofT cells which may cross-react with homologous peptides of exogenousorigin, all performed in a non-transgenic system. The example emphasizesthe fact that without a method such as that of the current invention tocapture T cells, it is not possible to evaluate polyclonal antigenspecific chimeric T cell selection in a non-transgenic system. This isbecause conventional methods that examine cytokine presense or cellproliferation cannot identify cells specific for relevant antigen.

The self-peptide Iα52 (ASFEAQGALANIAVDKA) (Seq. Id. No. 1), used inthese experiments corresponds to residues 52 to 68 of the α chain of theI-E molecule. It represents one of the most abundant peptides naturallyprocessed and presented in the context of I-A^(d) (Hunt, et al. (1992),Science, vol. 256:1817-20; Rudensky, et al. (1991), Nature, vol.353:622-27). These experiments demonstrate that T cells specific for aparticular antigen can be identified from a polymorphic population ofcells. In addition, functional and genetic characteristics of the cellpopulation were demonstrated (Table 1).

ITABLE I Phenotypic and Functional Characteristics of Thymocytes Beforeand After Fetal Thymic Organ Culture Day 6 of Day 16 FTOC+ of Day 6 of10⁻² mM Gestation FTOC Iα52 CD3^(lo)CD4+/CD8+6% 1.3% 1.1%CD3^(lo)HSA^(hi)99 3.9 4.1 CD3^(lo)CD69+2<1<1 CD3^(hi)<1 47 34CD4+CD8+CD3^(hi)HSA^(hi)<1 82 84 CD3^(hi)CD69+<1 10 11 Cell Number N/D1.55×10⁵# 1.65×10⁵# Iα52 N/D 0.5x±0.1 6.2x±2.7 Stimulation Index Hi15N/D 0.1x±0.06 2.5x±1.1 Stimulation IndexBALB/c embryonic thymus lobeswere made into single cell suspensions, and analyzed by flow cytometry.Cell numbers are an average of eight to ten thymus lobes from FTOC. Datain the table represent between two and four experiments. #Standarddeviations are 3.1×10⁴ and 1.5×10⁵ respectively.

Materials and Methods Antigens

The I-A^(d)-derived peptide Iα52 (ASFEAQGALANIAVDKA) (Seq. Id. No. 1)was synthesized (Research Genetics) using standard solid phase peptidesynthesis method. Peptides used for flow cytometry were biotinylated(b-peptides) using a kit (Sigma) and were separated from free biotin byHPLC.

Generation of Lymphocytes from Fetal Thymic Organ Cultures (FTOC)

BALB/c embryonic thymi were harvested day 16 of gestation and placedonto 0.4 um cell culture inserts (Fisher) in six-well plates containingRPMI 1640, 10% FCS, 1% P/S/G, with or without Iα52 (10⁻¹ mM to 10⁻¹⁰mM), at 37° C. and 6% CO₂ for six days. Cells were dissociated with aglass grinder (Kontes). Cell recovery was between 1.0×10⁵ and 2.8×10⁵cells/lobe. Post-FTOC cultures were performed using a 3:1 ratio ofirradiated BALB/cspleen cells, 20 U/ml rIL-2, 10 ug/ml Hi15 or Iα52, 10%FCS, 1% P/S/G, in RPMI 1640 at 37° C. and 6% CO₂.

Preparation of Liposome:MHC:Antigen Complexes

Liposomes were prepared as described in Example 1.

Flow Cytometry

Flow cytometry was performed as described in Example 1.

Results

It has been shown previously that different concentrations of the samepeptide have strong implications for T cell selection (see Sebzda, etal., Science, vol. 263:1615-18). We confirmed this phenomenon usingvarious concentrations of Iα52 peptide to demonstrate its influence uponpeptide-mediated positive selection and to define, directly, the antigenspecificity of a positively selected T cell population in anon-transgenic BALB/c model.

FIG. 10 shows the influence of Iα52 on the maturation of antigenspecific thymocytes in fetal thymic organ cultures. FIG. 10(a)represents differing concentrations of Iα52 added to 6-day FTOC usingday 16 of gestation BALB/c embryonic thymus lobes. Thymocytes wereanalyzed by three color flow cytometry to determine the percentages ofCD3HI, CD4, and CD8 double positive and CD3HI CD4 single positive cells.With FTOC alone in the absence of any added peptide, 20.7% of CD4+ cellsbound liposome: Iα52:biotinylated-Iα52 complexes during one week. Thisfinding reflects the availability of Iα52 as an abundantly represented,naturally processed peptide available for thymic selection.

FIG. 10(b) shows VP analysis by FACS of cells taken directly from day 16of gestation embryonic thymus lobes, and from FTOC supplemented with10⁻² mM Iα52.

FIG. 10(c 1) represents thymocytes from FTOC without antigen, or withaddition of the Iα52 peptide (c2), which were analyzed by FACS usingpropidium iodide to measure cell viability. Analysis of CD4, CD8 ratiosfrom the two cell cultures (c3,4) is representative of those used in theabove titration.

Performance of the detection of antigen specific thymocytes by FACSusing anti-CD4 antibody and liposome: Iα52:b-Iα52 complexes bound tof-strep is shown in FIG. 11. When thymic lobes were cultured for oneweek without the presence of 10⁻² mM Iα52 (FIG. 11-1), a relativeincrease (to 50.9%, FIG. 11-2) of liposome:Iα52:biotinylate-d-Iα52, CD4+cells was seen in the presence of 10⁻² mM Iα52. These data demonstratefor the first time that a self-derived peptide can induce positiveselection in a non-transgenic system. As a control for antigenspecificity in the T cell capture assay, the unrelated, biotinylatedpeptide EBV1 (TRDDAEYLLGRESVL), (Seq. Id. No. 2) derived from the EBVprotein balf2 (residues 1030-1045) was used. EBV-1 binds efficiently toIα52 and is a good immunogen in adult BALB/c mice (La Cava et al,submitted). Biotinylated EBV1 peptide complexed to Iα52 in liposomesbound 0% of CD4+ cells from FTOC (FIG. 11-3).

EXAMPLE 3 Indentification of Cross Reactive T Cells with Specificity forHomologous Peptides

The method of the invention was used to further examine the capacity forclosely related antigenic moieties to cross-react, in this case usingthe murine model seen in Examples 1 and 2. Evidence of suchcross-reaction provides the basis for an explanation of some autoimmunedisease states. In this Example, the ability of T cells selected by theself-MHC-derived peptide Ia52 to cross-react with the homologous peptideof non-self origin Hi15, was examined by performing antigen-specific Tcell analysis.

Materials and Methods Antigens

The Iα52 derived peptide Iα52 was synthesized as described in Example 2.Hi15 (TSFPMRGDLAKREPDK) (Seq. Id. No. 3) was synthesized by standardsolid phase peptide synthesis technique (Research Genetics). Hi15 wasidentified among 20 candidates with an arbitrary homology score of 20,based on homologies including potential MHC-binding residues. The searchwas performed on the non-redundant database scanned by Blast 2 program,available on the NCBI website. Peptides were >90% pure (ResearchGenetics). Peptides used for flow cytometry were biotinylated(b-peptides) using a commercial kit (Sigma) and were separated from freebiotin by HPLC. Experiments were performed to determine the possibleinterference of the biotin label on the specificity of T cell receptorsfor the Hi15 peptide using the post-synthesis biotinylated Hi15 andN-terminus biotinylated peptide separately complexed to MHC molecules inliposomes and labeled with different colors of conjugation tostreptavidin molecules. Results showed equal binding of the twopreparations to a T cell line having specificity for both Ix52, and Hi15 (not shown).

Generation of Lymphocytes from Fetal Thymic Organ Cultures (FTOC)

FTOC was prepared as described in Example 2.

Preparation of Liposome:MHC:Antigen Complexes

Liposomes were prepared as described in Example 1.

Flow Cytometry

Flow cytometry was performed as described in Example 1.

Results

These experiments show that a T cell population derived fromIα52-supplemented FTOC can be determined, characterized and expanded.Essentially, an artificial APC will comprise MHC:peptide complexesstabilized into liposomes, with the addition of transmembrane proteinswhich accomplish stabilizing, co-stimulatory, and/or modulatoryfunctions. These accessory and co-stimulatory molecules comprise, butare not limited to, any or all of the following:

-   -   (i) ICAM 1 as adhesion molecule to facilitate initial        interaction between the T cell and the APC.    -   (ii) anti-CD28 transmembrane antibody facilitates the        propagation of antigen-specific T cells isolated by T cell        capture using artificial APCs such that up to 20 replicative        cycles have been obtained, which represents a valid alternative        to T cell expansion and cloning using autologous APC systems,        often an insurmountable hurdle in human systems.    -   (iii) B7-1 maybe used instead of anti-CD28. The cells obtained        from this treatment may exert immunoregulatory function in        autoimmunity.    -   (iv) B7-2 may be used instead of anti-CD28. Cells obtained from        this treatment may have immunomodulatory properties in settings        such as cancer or infectious disease.

All of the aforementioned molecules are transmembrane proteins which canbe incorporated into liposomes according to above examples, or as analternative, bound to a solid support as in above examples.

Isolation and Immunomodulation of Antigen Specific T Cells

Antigen-specific T cells isolated according to above examples areincubated with the appropriate variant of the artificial antigenpresenting cells according to the desired objective, i.e. expansion,functional phenotype switch, etc. Day 16 of gestation BALB/c embryonicthymus lobes were harvested and cultured on 0.4 um filters in mediumsupplemented with 10-2 mM Iα52. Lobes were dissociated and cultured withautologous, irradiated APC, IL-2 and the Hi15 peptide. After two weeksof incubation with 10 ug/ml of Hi15, FACS analysis showed that 52.8% ofcells bound anti-CD4 and the self-derived Iα52:Iα52 peptide complexes(FIG. 12-1). The liposome: Iα52:biotinylated Iα52+:CD4+ cell complexeswere sorted and depleted of their liposome:I-Ad:Iα52+ components byincubation at 4° C. for thirty minutes followed by centrifugation at325×g for 10 minutes through 100% FCS (FIG. 12-2). The sorted cells werethen restained with liposome:I-A^(d) and the exogenous, biotinylatedpeptide Hi15 that was complexed to a streptavidin molecule conjugated toa fluorochrome. The restained T cells were then reanalyzed bythree-color flow cytometry using anti-CD4antibody,liposome:I-A^(d):b-Hi15 complexes, and liposome:I-A^(d):b-Iα52 complexesbound to different color f-strep molecules. Results showed that 52% ofthe cells tested positive (FIG. 12-3). To further demonstrate theability of TCRs selected by Iα52 to recognize the exogenous antigenHi15, T cell populations derived from Iα52 FTOC were expanded with Hi15and incubated with anti-CD4 antibodies and liposome:I-A^(d) complexesbearing either biotinylated-Iα52 or Hi15 bound to streptavidin moleculeslabeled with different fluorochromes. The results indicated that 31.1%of the CD4+ cells were positive for both peptides (double positive)(FIG. 12-4). The results of this Example 3 show that positive selectionby a self-peptide generates CD4+T cells that can recognize a homologous,exogenous peptide.

EXAMPLE 4 Identification of TCR Usage by Antigen Specific T Cells

Variable regions of TCRs on the cross-reactive cells identified inExample 3 were evaluated in order to demonstrate the absolute ability ofthe method of the current invention to evaluate TCR specificity andpotential cross-reactivity seen at a molecular level.

Materials and Methods Antigens

The Iα52 (ASFEAQGALANIAVDKA) (Seq. Id. No. 1) peptide (ResearchGenetics) and the Hi15 (TSFPMRGDLAKREPDK) (Seq. Id. No.3) peptide(Research Genetics) were >90% pure. Peptides used for flow cytometrywere biotinylated (b-peptides) using a commercial kit (Sigma) and wereseparated from free biotin by HPLC.

Generation of Lymphocytes from Fetal Thymic Organ Cultures (FTOC)

FTOC were prepared as described in Example 2.

Preparation of Liposome:MHC:Ag Complexes

Liposomes:MHC:antigen complexes were prepared as described in Example 1.

Flow Cytometry

Flow cytometry was performed as in Example 1.

Analysis of T Cell Receptor Gene Usage

To analyze the TCR-Vβ repertoire, 500 FACS-sorted cells were washedtwice and poly (A) mRNA was isolated with the Invitrogen Micro FastTrack mRNA Isolation kit (Invitrogen, San Diego). The resulting mRNA wasquantified with DNA DipStick kit (Invitrogen) and equal amounts of mRNAfrom each source were reverse transcribed using Invitrogen's cDNACyclekit. The cDNA was then submitted to a first PCR by using specific Vβsense primers and a common Cβ antisense primer (Lessin, et al. (1991),J. Invest. Dermatol, vol. 96:299-302). PCR conditions were 200 uM dNTPs,1× Taq polymerase buffer, cDNA, 20 uM each primer, 0.6 U Taq DNApolymerase (Boehringer Mannheim) in a 25 ul total reaction volume. After3 minute hot start at 94° C. and addition of Taq DNA polymerase at 80°C. using a Perkin Elmer 2400 thermal cycler, reactions were cycled 40times consisting of 30 seconds at 94° C., 30 seconds at 55° C. and 40seconds at 72° C., last extension 6 minutes. Five microliters were usedas template for a second PCR, whose conditions were identical to theprevious PCR, except that reactions were cycled 35 times and 0.125 U ofTaq DNA polymerase was used. Twenty-five microliters of the resultingPCR products were then analyzed by electrophoresis through a 4% agarosegel and ethidium bromide staining. DNA sequencing was performed at TheScripps Research Institute Core Facility using an ABI system.

Results

T cell lines obtained from either FTOC supplemented with 10⁻² mM Iα52,or from peripheral lymph nodes of animals immunized with Iα52, weresubsequently expanded in the presence of Hi15 and IL-2. Cells weresorted by FACS, as described in Example 1 using cytometry based onanti-CD4, liposome: Iα52:biotinylated-Hi15 andliposome:Iα52:biotinylated-α52 triple binding. Then, RT-PCR specific forthe known TCR Vβ families was performed.

The results of these experiments showed almost exclusive use of Vβ1 bycells that recognized both the selecting self-peptide Iα52 and thecross-reactive, non-self homologue Hi15 (Table II). We also obtained DNAsequences from three representative clones. This analysis showed onlymodest differences in amino acid sequence for various representativeclones, which all utilized Vβ1 genes (Table II). Molecular modelingshowed that these amino acid sequences had little effect on thepredicted conformation of the Vβ chain used by the different clones(FIG. 9).

TABLE II CDR3 Sequence Comparison and Variable Region Gene Usage of Iα52and Hi15 Cross-Reactive T Cell Receptors T Cell Isolated VDJ Linesequences R3F17 LHISAVDPEDSAVYFCASSQEFFSSYEQYFGPGTRL* R3F16 I R3F15 TVariable Region (Vβ) Gene Usage F7d 1, 8 Lines were generated fromsingle cell sorting by flow cytometry using the T cell capture assay.Detection in the assay was based upon I-Ad/b-Iα52 and I-A^(d)/b-Hi15double binding. These sequences contain the Vβ1 region of murine TCR inpositions 78-107. *Seq. Id. No. 4.

EXAMPLE 5 Identification, Isolation and Characterization of T CellsSpecific for Rheumatoid Arthritis-Related Antigens

Initiation of autoimmune diseases such as rheumatoid arthritis isthought to be dependant on the recognition by T cells of one or more“pathogenic” antigens which may be responsible for triggeringproinflammatory events which lead to chronic autoimmune damage. Severaldifferent hypotheses and experimental approaches have led to theidentification of a pool of potential pathogenic antigens.

In this Example 5, it is demonstrated how T cells specific forrheumatoid arthritis-related antigens can be identified and enumeratedstarting from polymorphic T cell populations with very diversespecificities. Peripheral blood mononuclear cells (PBMC) from a patientwith rheumatoid arthritis are incubated with a peptide called dnajpl(U.S. Pat. No. 5,773,570) corresponding to certain positions of the E.coli heat shock protein dnaJ. Certain peptides derived from dnaJ arehomologous to rheumatoid arthritis-associated HLA alleles, and have beenshown to be immunogenic in patients with rheumatoid arthritis. (Albaniet al., “Positive selection in autoimmunity: Abnormal immune responsesto a bacterial dnaJ antigenic determinant in patients with earlyrheumatoid arthritis,” Nat. Med. 1:448-452 (1995); Albani & Carson, “Amultistep molecular mimicry hypothesis for the pathogenesis ofrheumatoid arthritis,” Immunology Today 17:466-470 (1996); La Cava etal., “Genetic bias in immune response to a cassette shared by differentmicroorganisms in patients with rheumatoid arthritis,” J. Clin. Invest.100:658-663 (1997). Each of the above references are herein incorporatedby reference).

Materials and Methods Preparation of DNA

Genomic DNA is prepared from white blood cells by methods known to thoseskilled in the art utilizing ammonium acetate and isopropyl alcoholprecipitation with resuspension in TE (1 mM Tris, 0.1 mM EDTA, dH20, pH7.5).

PCR Amplification

DNA is enzymatically amplified by the polymerase chain reaction andamplification primers that have previously been reported for DQA1 andDQB1, for level I DRB typing, for level 2 allele assignment of DRB1specificities associated with DR52, and for DR4 and DR1 (Vooter, C. E.Tissue Antigens. 51:1 :80-87.; Barnardo, M. C. Tissue Antigens.51:3:293-300.; Mitsunaga, S. Eur J Immunogenet. 25:1:15-27.). GenomicDNA (5-10 ul at 80-150 ug/ml) is added to PCR mix consisting of 5 ul of2 mmole/ul dNTPs, 3 ul of each primer at 10 pmole/ul, 5 ul of 10×PCRbuffer, 0.5 ul of Taq polymerase (5 U/ul) and 23.5 ul sterile dH2O.Total reaction volume is 50 ul. DNA is amplified for 30 cycles in a DNAThermal Cycler (Perkin Elmer Cetus) with denaturation, annealing, andextension parameters that vary depending upon the locus studied. Ingeneral, annealing is at 53° C. for DQ and DRB level I, and at 60° C.for DRB1-specific families. Positive and negative controls are includedwith each run and care is taken to avoid any source of contamination.

SSOP Hybridization and Detection

After amplification, 5.0 ul samples (10% of total PCR mixture) are runon a 1.0% agarose gel to verify sufficient quantity of amplifiedproduct. The remaining portion of the samples (90% of total PCR mixture)are denatured prior to blotting and hybridization usingchem-luminescence using methods known to those skilled in the art.Membranes are washed twice with 2.times.SSC+0.1% SDS (0.3 M NaCl, 0.03 MNa-citrate, 0.1% SDS, dH2O, pH 7.0) for five minutes at roomtemperature, and then twice with 3 M TMAC buffer in the appropriatetemperature for the length of the probe. Fifteen-base pair probes arewashed at 50-52° C. and 18-base pair probes are washed at 59-61° C.Membranes are wetted with Lumi-Phos (Boehringer Mannheim), sealed inacetate sheets, and exposed to X-ray film for 1 to 5 minutes. Resultsare graded using 11th International Workshop criteria as follows:1:negative (definite) 2:negative (probable) 4:indefinite 6:positive(probable) 8:positive (definite) 9:positive (definite, more than doubleintensity). If unique hybridization patterns are found in the course ofthese studies sequence analysis is done. The patient employed in theexample was HLA typed by PCR.

HLA Purification

Lymphoblastoid cell lines from RA patients are used as a source of HLAmolecules, as known to those skilled in the art. Allele andspecies-specific monoclonal antibodies, whose corresponding hybridomasare already available, are produced as ascites liquid from BALB/c miceand purified on protein A, and covalently bound to a solid support(AffiGel, Bio-Rad, Richmond, Calif.). HLA molecules are purified byimmunoaffinity chromatography from lysates of 10⁸ lymphoblastoid cells.The yield ranges from 1 to 4 mg/preparation. Purity is assessed bySDSIPage and Western blotting. Molecules in an elution buffer containing0.2% octasilglucoside are stable for more than 4 months at 4° C.

Preparation of Liposome:MHC:Antigen Complexes

Liposomes are prepared as described in Example 1.

Flow Cytometry

Flow cytometry is performed as described in Example 1.

Results

T cells specific for the various combinations of the HLA, DR/DQ, and S1or dnaJp1 peptides were enumerated. T cells binding the S1 or dnaJp1peptides in the context of HLA DR are more abundant (FIGS. 13A-E). Seenin the figure are PMBC from an RA patient expressing disease associatedalleles DRB*10401 and DQ*30301 captured with DQ/P1 (FIG. 13-A), DQ/S1(FIG. 13-B), DR/PI (FIG. 13-C), DR/SI (FIG. 13-D), and DQ/Mtd1 (FIG.13-E).

In order to demonstrate cross-reactivity, T cells specific for thevarious HLA/peptide combinations are sorted and RT-PCR for TCR Vβ geneusage is performed. The results, shown in Table III, demonstrate that Tcells specific for the self-HLA derived peptide S1 cross-react with thehomologous peptide dnaJp1. Identification and isolation of T cells witha high pathogenic potential, as described in this Example 5 allowsmanipulation ex vivo to induce tolerization.

3TABLE III TCR Vβ Usage of T cells Isolated by Detection with RAAssociated HLA Molecules Bound by Self or Exogenous Peptides. HLA DQ HLADR dnaJP1 1, 8, 12 8, 14 S1 4, 12, 21 1, 9, and 14.

Peripheral blood mononuclear cells were isolated from an RA patientexpressing the RA associated HLA alleles DRB*10401 and DQB*30301. Thecells were cultured for five days in the presence of IL-2 and thebacterial heat shock protein peptide dnaJP1 then sorted by flowcytometry using HLA/Antigen combinations complexed into liposomes.RT-PCR was performed on sorted cells using primers specific for theknown Vβ regions. Data are representative of two separate experiments.

The numbers in the above Table III show that of the 23 genes of the Vβgene family, crossreaction occurs with only a few even though the familyis higly related. Additionally, the results show that individual membersof the family can be detected using the T cell capture method of theinvention using artificial APCs.

EXAMPLE 6 Identification, Isolation, and Characterization of T CellsSpecific for Juvenile Dermatomiosytis-Related Antigens

Juvenile Dermatomiosytis is a chronic autoimmune disease of unknownetiology. It has been reported that in several patients there is anassociation between relapse of the disease and documented Streptococcuspyogenes infection (Martin, A., Ravelli, A., Albani, S., J. Peds,121:739-742 1992). Sequences shared between Streptococcus M5 protein andthe human skeletal myosin, the target of the autoimmune process havebeen reportedly identified. It has also been demonstrated that theshared sequences contain epitopes which elicit cross-reactive T cellresponses. In this Example 6, it is demonstrated that the homologoussequences are actually recognized by T cells which bear the same TCR Vβgene.

Materials and Methods

Sequence-specific oligonucleotide probe HLA typing was performed asdescribed in Example 5.

Preparation of Liposome:MHC:Antizen Complexes

Liposome:MHC:antigen complexes are prepared as described in Example 1.

Flow Cytometry

Flow Cytrometry is performed as in Example 1.

Two alternative methods for visualization of the antigen-specific Tcells were employed. In the first method, affinity-purified HLAmolecules were stabilized by incorporation within the interior ofliposomes using a method similar to that described in Examples 1-5 andthen incubated with biotinylated peptides which were labeled using amethod similar to that described in Examples 1-5. In a second alternatemethod, a label, in this case fluorescent (FITC), was incorporateddirectly into the liposomesthemselves. Thus, the liposomes, rather thanthe peptides were labeled. FIGS. 14A-D shows data representing PBMC froma patient with JDM capture with a class I HLA and M61 -1, M61-1*, M61-2,and M61-2* (FIGS. 14-A to 14-D respectively).

Results from these experiments show that T cells which recognizehomologous peptides of either self or bacterial origin use the same TCRVβ genes, confirming cross-reactivity at a molecular level. In addition,a direct identification and enumeration of CD3+ antigen-specific T cellsis possible without detectable differences in sensitivity between thetwo labeling methods used. These results also suggest that directincorporation of a label, e.g., FITC, into the liposomes themselves, orincorporation of a labeled (e.g., biotinylated) transmembrane proteinwhich may or may not interfere with the TCR:antigen:MHC interactions,represent valid methods as alternatives in labeling of the liposome.This may be advantageous in that a label (e.g., a biotin molecule) whencomplexed with the peptide, may interfere with the interactions amongagretopes and epitopes within the TCR:antigen:MHC complex. This may beespecially true with class I MHC molecules, in which the antigen bindinggroove is open only at one end. Our diverse approaches bypass thesepotential limitations.

EXAMPLE 7 Identification and Enumeration of T Cells Specific forPeptides from Pathogenic Organisms

Physiologic, as well as vaccine-induced, immune responses to infectiousagents are based on T cell recognition of, and reactivity to,immunogenic epitopes of the infectious agents. In several instances,immunogenic epitopes of microorganisms employed in vaccines, and theirHLA restrictions, have been identified. One example is the influenzavaccine.

In this Example, quantitative and qualitative T cell responseabnormalities in response to influenza vaccination can be identified.Peptides encompassing the major epitopes of the influenza virus areemployed for analysis. Antigen specific T cells are identified andisolated as described in Example 1. The population to be screened iscomprised of elderly persons who are vaccinated and then screened forimmune responses. Antigen specific T cells are quantified and isolatedfrom both the responder and non-responder groups. Phenotypical andfunctional characteristics are then evaluated, and the results comparedbetween the two groups to analyze whether hyporesponsiveness in some ofthe vaccinated subjects is related to lack of specific T cells.

PBMC are incubated with the relevant and control peptides for five daysin order to increase precursor frequency. Cells are then incubated withHLA-peptide complexes, and positive cells are enumerated by FACS.Commercially available cell lines are the source of soluble HLAmolecules (Corriel Cell Repository).

Antigen specific T cells are evaluated for the production of cytokineswhich are related to either effective responses to viruses (IFN, IL-2)or associated with anergy (as in the case of using, for example, IL-10).This analysis elucidates differences, if any, between antigen specific Tcells of responders versus non-responders. Hence, important functionaldifferences are evaluated at an antigen-specific T cell level. Membranephenotype of the isolated antigen specific T cells is evaluated, withparticular attention to early activation markers, such as CD69, andmemory, such as CD45RO. This test helps distinguishing anamnestic fromrecently induced responses. mRNA is purified and RT-PCR using Vβ chainfor the T cell receptor specific primers is performed. The definition ofthe T cell receptor (TCR) used by the various subjects may help inpointing out differences between responders and non-responders due togenetic differences in the TCR repertoire. This shows the potential forthe technology of the current invention to discriminate betweenanamnestic and recall immune responses at the level of antigen-specificT cells

Materials and Methods Lymphocyte Proliferation Assays

Preliminary experiments are performed to identify the optimal conditionsfor culture. Initially, cell cultures are performed in duplicate forthree to seven days, using 5 million cells/well. (All antigens haveshown a range of optimal responses between 1 and 10 ul/ml). Theproliferation assays are conducted as controls to compare against the Tcell capture specificity of the invention.

Lymphocyte Cytotoxicity Tests

Cytotoxic responses are measured using a LDH-release kit (Promega). Thetests are performed according to manufacturer's instructions. Effectorsare incubated for 5 days with IL2 and the relevant peptides or withirrelevant antigens. Targets are irradiated autologous PBMC or, whenavailable, EBV-transformed lymphoblastoid cell lines. Targets are pulsedwith the relevant antigens overnight. The cytotoxicity assays areconducted as controls to compare against the T cell capture specificityof the invention.

Antigens

Synthetic peptides (Matrix 58-66:GILGFVFTL (Seq. Id. No. 5);Nucleoprotein 82-94:VKLGEFYNQ (Seq. Id. No. 6)) are purchased fromResearch Genetics.

MHC Purification

Commercially available lymphoblastoid cell lines (Corriel CellRepository) are used as a source of MHC molecules and purified byimmunoaffinity chromatography using anti-HLA class I antibodies in amanner similar to that shown in Example 5.

Liposome:MHC:antigen complexes are prepared as described in Example 1.Antigen specific T cells are prepared as described in Example 1.Intracellular immunofluorescence staining of cytokines was performed asdescribed in Example 10.

Cytokine Measurement by RT/PCR

Messenger RNA is extracted from approximately 7×10⁶ cells by usingOligotex Direct mRNA Kit (Qiagen, Chatsworth, Calif.). mRNA isreverse-transcribed into cDNA with the oligo dT primer (RT-PCR Kit,Stratagene, La Jolla, Calif.). Two ul of single strand cDNA areamplified using the cytokine specific forward and reverse primer sets,IL-2, IL4, TNF-α, INF-γ. Quantitative measurement of various cytokinesusing competitive PCR is performed (Biosource reagents). In this method,a known copy number of an exogenously synthesized DNA used as aninternal control sequence (ICS) is mixed with the sample prior toamplification. The ICS is constructed to contain identical primerbinding sites as the cytokine to be analyzed, and a unique binding sitethat allows the resulting amplicon to be distinguished from the cytokineproduct. Detection of the amplification product is by non-radioactivemicroplate techniques.

T Cell Receptor Analysis by PCR

Messenger RNA is extracted from a minimum of 70 cells by usingMicro-FastTrack Kit (Invitrogen, Carlsbad, Calif.). The yield of mRNA isabout 2 ul that is resuspended into 20 ul of water. Two ul of mRNA foreach reaction is reverse-transcribed into single strand cDNA with theoligo dT primer (cDNA Cycle Kit, Invitrogen, Carlsbad, Calif.). 1.5 ulof single strand cDNAs are amplified with constant primer and various Vregion primers (Vb1-Vb24), the sequences of which may be designed bythose knowledgeable in the art. For example, 20 pico moles of eachprimer and 1.25 units of Taq polymerase (Boehringer Mannheim, Germany)are used. The total volume is 25 ul. The cycling parameters for PCR isindicated as: heat PCR reaction mixture without Taq and dNTPs at 94° C.for 4 min, then perform 40 cycles of 30 seconds at 94° C., 30 seconds at58° C. and 30 seconds at 72° C. The final elongation is 7 minutes at 72°C. Add 0.5 ul of 100 mM dNTPs and 0.25 ul of Taq Polymerase (1.25 units,Boehringer Mannheim) at end of 58° C. of first cycle. The PCR-amplifiedproducts are analyzed on 4% agarose gel.

In summary, the experiments described in this Example 7 are accomplishedaccording to the following steps:

-   -   (i) MHC typing by PCR of the test sample derived from a test        subject, (e.g. a patient that is to receive treatment against an        infectious agent);    -   (ii) Purification by immunoaffinity chromatography or by other        methods known to those skilled in the art, of MHC molecules        corresponding to that of the test subject's. The source for        these molecules may be directly from an individual or from cell        lines homozygous for the HLA alleles.    -   (iii) Synthesis of liposomes containing MHC:peptide complexes.        (These liposomes may be tagged according to any of the methods        described herein or known by those in the art.);    -   (iv) Incubation of PBMC from the test subject with the        MHC:peptide tagged liposomes, and;    -   (v) counting of the binding T cells for the purpose of        determining which peptides have bound thereby indicating the        specific peptides effective in binding antigen-specific T cells.

Methods described in this Example are useful in determining the numberof vaccine-specific T cells in peripheral blood, both in normal andimmune compromised individuals, for example, as a tool for decisionsregarding the opportunity for vaccination in immune compromisedsubjects, and in determining the efficacy of vaccination, measured asincrease in the number of vaccine-specific T cells after vaccination.

EXAMPLE 8 Diagnosis and Immunomodulation of Allergic Disease

Allergy is mediated by release of pro-inflammatory and vasoactivemediators triggered by binding of allergen specific immunoglobulins withreceptors of inflammatory cells at the site of allergen contact withcellular tissues. The production of allergen specific immunoglobulinsappears to be mediated by strong interactions between B and T cells, anexample being Casein in the pathogenesis of lactose intolerance (Albani,S. Annals of Allergy. 63:12:489-492.). It is therefore of importance tohave the possibility to identify allergen specific T cells, isolate themand manipulate them ex vivo. An outline of the strategy to be employedis:

-   -   (i) MHC typing of the test subject by PCR technology;    -   (ii) Identification of candidate “allergenic” peptides, based on        current knowledge of the field. (When a candidate epitope on a        given proteic allergen will not be already available,        computerized analysis of MHC binding motifs will enable the        identification of candidate peptides to be used.)    -   (iii) Synthesis of liposomes containing relevant MHC:peptide        complexes. (Such liposomes can be tagged using any of the        techniques described herein or known by those in the art.);    -   (iv) Incubation of PBMC of the test subject with the        MHC:peptide:tagged liposomes complexes;    -   (v) Identification and enumeration of allergen specific T cells;    -   (vi) manipulation ex vivo of such cells, by stimulation in        culture with the antigenic peptide in the presence of stimuli        related to induction of TH-1 phenotype

Materials and Methods

Lymphocyte proliferation assays are performed as described in Example 7.

Lymphocyte cytotoxicity tests are performed as described in Example 7.

Antigens

Peptides are identified based on scanning the sequences of the proposedallergens for MHC-binding motifs and synthesized according to standardpeptide synthesis methods know to those in the art.

MHC purification is performed as described in Example 7.

Liposome MHC:antigen complexes are prepared as described in Example 1.

T cells, generated as described in Example 1, are captured by separationusing flow cytometry as described in Example 1.

Intracellular immunofluorescence staining of cytokines, is performed asdescribed in Example 5.

Cytokine measurement by RT/PCR is performed as described in Example 7.

T cell receptor analysis by PCR is performed as described in Example 7.

This Example therefore shows that artificial APCs may be used todiagnose and monitor progress of therapies and modulation level ofspecific responses in a patient's T cells.

EXAMPLE 9 Identification of Cancer-Specific T Cells Epitopes

It is commonly accepted that lack of immunity to cancer (e.g.,melanoma), depends on low antigenicity of the neoplasm, as well as onfunctional and/or numerical deficiencies of antigen-specific T cells.

Several therapeutical approaches are currently underway to improve theefficiency of recognition of and reactivity to cancer antigens by Tcells. Unfortunately, the efficiency of the treatment can be measured,to date, only in terms of clinical outcome. This Example 9 describes asolution to this problem, by enabling identification, enumeration,characterization and possible manipulation of cancer-specific T cells.Hence, efficiency of the treatment will be measured in terms of increasein antigen-specific precursor frequency, and also in terms of functionaloutcome of antigen recognition. This latter aspect is of particularimportance in those instances where the therapy is aimed at activationof otherwise dormant T cells.

-   -   (i) The following protocol is applied:

MHC typing by PCR of the test subject;

-   -   (ii) Identification of candidate peptides. For example, sources        for antigen in the treatment of melanoma include MAGE-1, MAGE-3,        MART-1/melan-A, gp100, tyrosinase, gp75, gp15, CDK4 and        beta-catenin. When a candidate epitope on a given protein        allergen will not be already available, computerized analysis of        MHC binding motifs will enable the identification of candidate        peptides to be used;    -   (iii) Synthesis of liposomes containing relevant MHC:peptide        complexes;    -   (iv) Incubation of PBMC of the test subject with the        MHC:peptide:tagged liposomes complexes;    -   (v) Identification and enumeration of antigen specific T cells.

Materials and Methods

Lymphocyte proliferation assays are performed as described in Example 7.

Lymphocyte cytotoxicity tests are performed as described in Example 7.

Antigens

Synthetic peptides will be identified based on scanning the sequences ofthe proposed allergens for HLA-binding motifs.

MHC Purification

MHC purification is performed as described in Example 7. Commerciallyavailable lymphoblastoid cell lines (Corriel Cell Repository) are usedas a source of MHC molecules, and purified by immunoaffinitychromatography using anti-MHC class I antibodies, available in ourlaboratory.

Liposome MHC:antigen complexes are prepared as described in Example 1.

T cells, generated as described in Example 1, are captured by separationusing flow cytometry as described in Example 1.

Intracellular immunofluorescence staining of cytokines, is performed asdescribed in Example 5.

Cytokine measurement by RT/PCR is performed as described in Example 7.

T cell receptor analysis by PCR is performed as described in Example 7.

As shown, a peptide based method for monitoring cancer therapy isestablished using antigen-specific artificial APCs. This methods allowsfor studying the progress of the therapy and the state of the cancer.

EXAMPLE 10 Methods for Distinguishing “Bystander T Cells” fromAntigen-Specific T Cells

The experiments described in this Example demonstrate thatantigen-specific T cells can be identified and enumerated anddistinguished from antigen non-specific T cells present in the sametissue. These latter cells (so-called “bystander T cells”) mayparticipate in pathogenic processes. This has particular relevance forautoimmune and allergic diseases, where the initiating event may be therecognition of a pathogenic antigen by a specific T cell population.This interaction leads to production of pro-inflammatory or pro-allergicmediators (i.e., cytokines) by the antigen-specific T cells. Thecytokine cascade will subsequently involve T cells that are not specificfor the antigen (bystander T cells), which may then participate in thepathogenic processes, amplifying significantly the degree of the damage.It is important in a clinical or research setting to discriminatebetween the antigen-specific and bystander populations in order toevaluate, for instance, the efficacy of a treatment. The strategy may besummarized as follows;

-   -   (i) Identification of candidate peptides, based on current        knowledge of the field. When a candidate epitope on a given        protein antigen is not already available, computerized analysis        of MHC binding motifs will enable the identification of        candidate peptides to be used in the “T cell capture”. Such        analysis is standard practice to those skilled in the art;    -   (ii) Synthesis of liposomes containing relevant MHC:peptide        complexes. Such liposomes can be tagged using any of the        techniques described herein;    -   (iii) Incubation of PBMC of the test subject with the        MHC:peptide:tagged liposomes complexes;    -   (iv) Identification and enumeration of antigen specific T cells.

Materials and Methods Lymphocyte Proliferation Assays

Preliminary experiments may be performed to identify the optimalconditions for culture. Cell cultures may be performed in duplicate forthree to seven days, at 5 million cells/well. All antigens have shown arange of optimal responses between 1 and 10 ul/ml.

Lymphocyte Cytotoxicity Tests

Cytotoxic responses may be measured using a LDH-release kit(commercially available). The tests may be performed according tomanufacturer's instructions. Effectors may be incubated for 5 days withIL2 and either the relevant peptides or with irrelevant peptideantigens. Targets may be irradiated autologous PBMC or, when available,EBV-transformed lymphoblastoid cell lines. Targets may be pulsed withthe relevant antigens overnight.

Antigens

Synthetic peptides are identified based on scanning the sequences of theproposed allergens for MHC-binding motifs.

MHC Purification

Commercially available lymphoblastoid cell lines (Corriel CellRepository) may be used as a source of MHC molecules and purified byimmunoaffinity chromatography using anti-MHC class I antibodies.

Preparation of Liposome MHC:Ag Complexes

Preparation of APCs are the same as that performed for Example 1.

FACS

A Beckton Dickenson FACS Star with LYSIS II software was used tovisualize cells. Sortings were performed using standard procedures.

T Cell Capture

T cells, generated as described, are captured by separation using flowcytometry. Bulk sorted cells are either cultured as described,immediately used for DNA analysis, or used directly for reanalysis usingFACS. Yields from bulk sortings range from 2000-16,000. Single cellsorts utilized 96 well culture plates containing media previouslydescribed. Fresh irradiated APCs are added to the single cell culturesonce per week, at which time analysis of clonal expansion is performed.Of 96 wells of sorted “events”, generally 8-12 show good expansion overa six week period.

Intracellular Immunofluorescence Staining of Cytokines

Human PBMC are isolated by density centrifugation and stimulated for 36hours with peptides at 10 ul/ml in the presence of 2 uM monensin. Cellsare washed in PBS with 2% FCS and incubated with Fc-block for 5 min. at4° C. PE-conjugated anti-CD3 monoclonal antibodies (mABs) are added andcells are incubated for 30 min. at 4° C. Cells are washed, fixed 20 min.at 4° C., and resuspended in a solution containing either FITCconjugated anti-IFN-γ mAbs or FITC conjugated anti-IL4 or IL2 mabs.Cells are incubated for 30 min at 4° C., washed twice in PBS with 2% FCSand their fluorescence measured using a Becton Dickinson FACScan. Flowdata are analyzed using Lysis II software (Becton Dickinson).

Cytokine Measurement by RT/PCR

Messenger RNA is extracted from approximately 7×10⁶ cells by usingOligotex Direct mRNA Kit (Qiagen, Chatsworth, Calif.). The mRNA isreverse-transcribed into cDNA using oligo dT primer (RT-PCR Kit,Stratagene, La Jolla, Calif.). Two ul of single strand cDNA areamplified using the cytokine specific forward and reverse primer setsfor IL-2, IL4, TNF-α, and INF-γ. Quantitative measurement of the variouscytokines was carried out using competitive PCR (Biosource). In thismethod, a known copy number of an exogenously synthesized DNA (ICS) ismixed with the sample prior to amplification. The ICS has beenconstructed to contain identical primer binding sites as the cytokine tobe analyzed, and a unique binding site that allows the resultingamplicon to be distinguished from the cytokine product. Detection ofamplicons is allowed by non-radioactive microplate techniques.

T Cell Receptor Analysis by PCR

Messenger RNA is extracted from a minimum of 70 cells by usingMicro-FastTrack Kit (Invitrogen, Carlsbad, Calif.). The yield of mRNA isabout 2 ul that is resuspended into 20 ul of water. Two ul of mRNA foreach reaction is reverse-transcribed into single strand cDNA with theoligo dT primer (cDNA Cycle Kit, Invitrogen, Carlsbad, Calif.). 1.5 ulof single strand cDNAs are amplified with constant primer and differentV region primers (e.g., Vb1-Vb24). 20 pico moles of each primer and 1.25units of Taq polymerase (Boehringer Mannheim, Germany) were used. Thetotal volume is 25 ul. The cycling parameters for PCR are: heat PCRreaction mixture without Taq and dNTPs at 94° C. for 4 min, performanceof 40 cycles for 30 seconds at 94° C., 30 seconds at 58° C. and 30seconds at 72° C. The final elongation is 7 minutes at 72° C. Add 0.5 ulof 100 mM dNTPs and 0.25 ul of Taq Polymerase (1.25 units, BoehringerMannheim) at end of 58° C. of first cycle. The PCR-amplified productswere analyzed on 4% agarose gel. The sequences of Vβ-specific primersthat may be used may be designed by those knowledgeable in the art.

This example therefore shows that antigen-specific immunotherapy can beused to influence populations of T cells having different specificitythat participate in the pathogenic process.

EXAMPLE 11 Immunoaffinity Chromatography for Positive Selection ofAntigen-Specific T Cells

As demonstrated in this Example, MHC:antigen complexes may beincorporated into liposomes as described in certain embodiments of theinvention above and bound to a solid support. By orienting the complexesso as to optimize the chance of interaction between the T cell receptorand the complexes, antigen-specific T cells may be isolated frombiological samples.

Immunoaffinity Chromatography Columns

MHC:peptide complexes may be bound to hydrazide coated glass beads.Alternatively, molecules having affinity for irrelevant molecules may bebound to glass beads coated with a hydrazide linker and may be used tobind a irrelevant molecule-containing liposome:MHC:peptide complexes. Aslurry of either of the above coated bead solid supports is incorporatedinto a compartment of a column such as that shown in FIG. 8 in acontrolled fashion. Polymorphic T cell populations contained in a liquidmedium are then introduced into the column, and the mixture is incubatedat room temperature for 30 minutes, with gentle turning of the column. Tcells with irrelevant specificity are not bound by the slurry and willflow through. Peptide or antigen-specific T cells that bind duringmixing are then removed from the solid supports by incubating the columnat 4° C. for 30 minutes.

EXAMPLE 12 Ex Vivo Depletion of Cells Related to a Pathogenic Process:Antigen-Specific Leukapheresis

In certain situations, it is desirable to deplete living systems of Tcells having specificity for a given antigen. In the case ofautoimmunity or allergy, these T cells may be involved in pathogenicprocesses and their depletion may therefore induce clinical improvement.Likewise, in the case of transplantation, recognition of a donor'sepitopes and ideotopes as non-self will cause graft vs. host rejectionof a transplanted organ. In such instances, depletion of the recipient'sT cell population that recognize the foreign epitopes/ideotopes isbeneficial. Depletion of such reactive T cells may be accomplished byconnecting in line with the general circulation of a patient a devicecomprising MHC:antigen complexes bound to a solid support and orientedto optimize TCR:complex interaction. For example, MHC:peptide complexesmay be associated in liposomes containing irrelevant molecules which maythemselves be captured by glass beads coated with molecules havingspecificity for the irrelevant molecules. Appropriate filters,sterilization and heating procedures may be used in a manner similar tothat currently employed in conventional leukaphoresis procedures. Inoperation, whole blood or blood enriched for T cells is allowed to flowthrough the device. Antigen-specific T cells are bound and eliminatedfrom solution. The slurry is continuously stirred, and after appropriatetime periods portions of it are incubated at 4° C. in order to elute theantigen-specific T cells that have bound to the solid support associatedMHC:antigen complexes. For clinical conditions where suchantigen-specific T cells are harmful, such cells can be simplydiscarded.

The specific procedure with respect to autoimmunity comprises:

-   -   (i) Preparing a column in which the MHC:antigen:accessory        molecule:other molecule complex is designed to interact with T        cells which may be involved in the pathogenesis of autoimmune        desease using methods described in Example 11.    -   (ii) Allowing blood from the patient to flow through the column        containing. artificial APC prepared in step (i), by which means        antigen specific T cells will be retained.

The specific procedure with respect to transplantation comprises:

-   -   (i) Preparing a column in which the MHC:antigen:accessory        molecule:other molecule complex is designed to interact with T        cells which may be involved in either the graft vs. host disease        of bone marrow transplant or the pathogenesis of graft        destruction in allogenic solid organ transplant using methods        described in Example 11.    -   (ii) Allowing blood from the patient to flow through the column        containing artificial APC prepared in step (i), by which means        antigen specific T cells will be retained.

EXAMPLE 13 Ex Vivo Manipulation of Antigen Specific T Cells:Bidirectional Switch from Th1 to Th2-Type Functional Phenotype

In the case of infectious disease or cancer, it may be beneficial toisolate cells with a given antigen specificity in order to change theirfunctional phenotype, for example, by manipulating such cells using theartificial antigen presenting cells described in this Example.Manipulated cells can then be reintroduced for immunomoduation.

Antigen-specific T cells, isolated according to Example 1 or Example 2,are incubated with the appropriate variant of the artificial antigenpresenting cells according to the following examples.

Expansion of Antigen-Specific T Cells

Isolated cells (10,000/ml) are incubated in standard culture medium withAPC expressing the relevant MHC:peptide combination, ICAM1 as adhesionmolecule to facilitate initial interaction, and anti-CD28 transmembraneantibody. The latter is employed as a co-stimulatory molecule to induceT cell proliferation without affecting Th bias. This type of approach isthe antigen-specific equivalent of the recent approach at T cellexpansion using anti-CD3/anti-CD28 molecules. Up to 20 replicativecycles have been obtained in this system, which represents a validalternative to T cell expansion and cloning using autologous APCsystems, often an insurmountable hurdle in human systems.

Expansion and Immunomodulation of Cells from a Th1 to a Th2 FunctionalPhenotype

(i) In one expansion, B7-1 may be used as a co-stimulatory moleculeinstead of anti-CD28. The cells obtained from this treatment may exertan immunoregulatory function in the autoimmunity condition.

(ii) In another expansion, B7-2 may be used as a co-stimulatory moleculeinstead of anti-Cd28. Cells obtained from this treatment may haveimmunomodulatory properties in settings such as cancer or infectiousdisease.

Specifically, an artificial APC comprises MHC:peptide complexesstabilized into liposomes, with the addition of transmembrane proteinswhich accomplish co-stimulatory functions. The molecules may compriseany or all of the following:

-   -   (a) ICAM1 as adhesion molecule to facilitate initial interaction        between the T cell and the APC.    -   (b) anti-CD28 transmembrane antibody is employed as a        co-stimulatory molecule which may induce T cell proliferation        without affecting Th bias. This type of approach is the        antigen-specific equivalent of T cell expansion using        anti-CD3/anti-CD28 molecules. Up to 20 replicative cycles have        been obtained in this system, which represents a valid        alternative to T cell expansion and cloning using autologous APC        systems.    -   (c) B7.1 may be used instead of anti-CD28. The cells obtained        from this treatment may exert immunoregulatory function in        autoimmunity.    -   (d) B7.2 may be used instead of anti-CD28. The cells obtained        from this treatment may have immunomodulatory properties in        settings such as cancer or infectious disease.

All of the aforementioned molecules are transmembrane proteins which canbe incorporated into liposomes according to above examples, or as analternative, bound to a solid support as in above examples.

Isolation and Immunomodulation of Antigen Specific T Cells

Antigen-specific T cells isolated according to the above examples areincubated with the appropriate variant of the APC according to thedesired objective, i.e. expansion, functional phenotype switch, etc.

EXAMPLE 14 Monitoring Immunological Outcome of Intervention ofAntigen-Specific T Cells by Correlation of Clinical Outcome with T CellPhenotype

In this example, the invention is applied to evaluating the clinicaloutcome of treatment regimens by correlating the phenotype ofantigen-specific T cells with clinical outcome. Specifically, theinvention is applicable to clinical monitoring and clinical trials wherethere is a need to evaluate the effectiveness of artificial APC inducedT cell responses. The antigen-specific T cells associated with aresponse (or disease state) are identified followed by theidentification of their functional phenotype. The phenotype identifiedis then correlated and monitored against the progression of a patient'sresponse to various treatment regimens.

EXAMPLE 15 Interaction of Artificial APCs with T Cells to Induce Cappingof Transmembrane Proteins

The immune synapse is the cluster of transmembrane molecules whichensures specific interaction between antigen specific T cells andantigen presenting cells. The outcome of these interactions is antigenspecific response by the T cells, mediated by signaling through the TCR.Several factors contribute to significant quantitative and qualitativedifferences in the response provided by the T cell. These factorsinclude affinity of interaction between MHC and peptide, and between theTCR and the MHC/peptide complex, the number of moieties available forinteraction, and the relative concentration of the moieties availablefor interaction. In a physiological situation, the triggering number ofinteracting molecules is achieved by “capping”, a phenomenon, whichoccurs when transmembrane molecules are allowed to freely migrate todefinite zones of the membrane, usually upon initial interaction betweentwo of the ligands involved, in this case the TCR and the MHC/peptidecomplex. In order to emulate such physiologic mechanisms, we employedartificial APCs loaded with the combination IA^(d)/OVA, and hybridoma8DO as the specific T cells. To visualize free movement of the TCR inthe T cell membrane, we employed a system where FITC-conjugated choleratoxin, a molecule known to combine with the intracellular portion oftransmembrane proteins, is introduced into the T cells. We show here forthe first time that artificial APCs can effectively emulate thephysiologic interactions between T cells and APC, particularly withrespect to allowing migration of molecules whose proper density is anessential requirement to induce T cell activation. Our system has alsothe potential to be a tool to study physiological mechanisms of T cellactivation, and to manipulate the intensity and quality of T cellresponse. This could be accomplished by controlling the affinity of theinteraction, and by adding the proper co-stimulatory and adhesionmolecules to the artificial APC.

Approximately 100 MHC/peptide binding sites are available on eachartificial APC for interaction with a single T cell, increasingtherefore the likelihood that artificial APCs will engage, throughmultiple interactions, low-affinity T cells. This may provide asignificant advantage over current methods for the identification of lowaffinity, class II restricted antigen-specific T cells, such as the onesoften involved in physiologic regulatory mechanisms or in diseaserelated autoimmune responses.

We first identified the optimal incubation times for the artificial APCwith the T cells, (i.e., about 20 minutes) at different stages ofevolution in the capping mechanism. Unlike systems where the interactingmolecules are fixed on planar membranes, interactions between T cellsand artificial APCs occur in culture medium, and rely on random movementfor molecular interactions. This results in the optimal period for aresult to be based on formation of “capping” of the TCR in the cellmembrane rather than just binding of the TCR and target moieties. In theexperiments shown in FIGS. 16-18, we incubated the artificial APC with8DO cells for 20 minutes, and evaluated the capping by confocalmicroscopy. In FIGS. 16A-D, we visualized comigration of the colera raftand the TCR, by incubating T cells with PE-conjugated monoclonalantibody (Pharmigen). TCR molecules are uniformely distributed in thecells membrane, as expected for cells, which are not interacting withstimulatory triggers. In experiments described in FIGS. 17A-D, wevisualized interaction of TCR by using the cholera toxin raft, and theMHC in the artificial APC membrane using a specific anti-MHC (Pharmigen)monoclonal, Alexa-red conjugated. In the field shown, the phenomenon ofprogressive migration of the TCR molecules toward the point of initialinteraction of the artificial APC with the TCR is evident at differentstages for different cells. In experiments described in FIGS. 18A-D, wevisualized the TCR using an Alexa red-conjugated anti CD3 monoclonal(source) and the liposomes using FITC-conjugated streptavidin whichbound to the biotin at the N-terminal of the OVA peptide. The results ofthis visualization procedure in FIG. 18 are overlapping with those shownin FIG. 17. Hence, we show here for the first time that artificial APCscan effectively emulate the physiologic interactions between T cells andAPCs, particularly with respect to allowing migration of molecules whoseproper density is an essential requirement to induce T cell activation.Our system also provides for studying physiological mechanisms of T cellactivation and the manipulation of the intensity and quality of T cellresponse. This “modulation” of T cell response could further beaccomplished by controlling the affinity of the interaction by addingthe proper co-stimulatory and adhesion molecules to the artificial APC.An additional preferred advantage of the system of the invention is thatit allows free movement of transmembrane proteins, enabling thesequential order of interactions among ligands, which occur upon firstcontact of the TCR with the APC in the immune system.

Immunohistochemistry Methods for FIGS. 16-18

Liposomes and 0.5×10⁵ cells were cytospinned at 300×g for 5 min onpoly-L-lysine coated slides (Sigma, St. Louis, Mo.). The cells werefixed for 10 minutes in 4% Paraformaldehyde, washed in PBS and thenincubated for 1 hours with the antibodies anti-CD3 IAd (Pharmingen, SanDiego, Calif.), at a concentration of 2-10 ul/ml in I-Block blockingbuffer (Tropix, Bedford, Mass.). Alexa 568 (red) species-specificsecondary antibodies (Molecular Probes, Oreg.) were used to visualizethe proteins. Coverslips were washed successively in PBS and deionizedH₂O for 5 min and mounted in Flouromount (Fisher, Calif.). Images wereobtained with a Zeiss Confocal Laser Microscope.

EXAMPLE 16 Identification of Class II Restricted T Cells in PolymorphicPopulations

Identification of antigen-specific, class II restricted T cells is muchmore difficult than the same identification analysis for class Irestricted T cells. These difficulties stem from the lower affinity ofinteraction of TCR/peptide/MHC for class II restricted responses, whencompared to class I. Known methodologies have identified only highaffinity antigen specific T cells based on soluble recombinant moleculesthat provide anywhere from one to four interaction sites for TCRbinding, wherein steric hindrance problems are thought to affect correctfolding of the recombinant molecules and their interactions with theTCR. In contrast, our results show that MHC/biotynilated peptidecomplexes can effectively visualize class II-restricted hybridoma Tcells regardless of affinity.

To evaluate the efficiency of our artificial APC system to identifyClass II restricted antigen specific T cells, we immunized BALB/c micewith the peptide Iα52. Iα52, is a naturally processed, abundantlypresented IE^(d)-derived peptide, which has previously been described asantigenic in BALB/c mice. Cells from regional draining lymphnodes, bothfrom AFA only and Iα52 immunized mice, were harvested. These cells werethen incubated with artificial APC presenting IA^(d)/α52 complexes. Asshown in FIG. 25B, 5.4% of CD4+T cells were Iα52 specific, while incomparison, only 0.7% CD4 cells were specific for the peptide in theIFA-only immunized mice (FIG. 25A). These data related well toantigen-specific T cell proliferation, measured by standard thymidineincorporation assay (FIG. 26).

Capture of T Cells by Artificial APCs is Effective in IdentifyingPolyclonal Class II Restricted Human T Cells

To identify human antigen-specific T cells, we employed as modelantigens a system comprising Pan-DR binder peptides (PADRE) ofcomparable affinity, and the influenza hemoagglutinin HA peptide. Thepeptides used were pan-DR binding peptide 965.10 PADRE (K(X)VAAWTLKAASeq. Id. No. 7), HA 307-319 (PKYVKQNTLKLAT Seq. Id. No. 8), and IAdbinding peptide OVA³²³⁻³³⁹ (ISQAVHAAHAEINEAGR Seq. Id. No. 9). Peptideswere synthesized as C-terminal amides, purified by reversed-phase HPLC,and checked by fast atom bombardment mass spectrometry. For use inMHC-binding assays and the T cell capture, peptides were biotinylatedduring peptide synthesis (only one biotin molecule, at the n-terminus,100% biotinylation).

The choice of such antigens was based on the concept that a high numberof polyclonal T cells could be found in PBMC if a peptide with highaffinity would be employed in the assay. We first defined the optimalmolar concentration to employ in loading PADRE pan-DR peptideKXVAAWTLKAA Seq. Id. No. 7, and showed the specificity of interactionbetween the PADRE peptide and the HLA molecules. T cells from a normalHLA DRB1*401 donor were first tested for the number of PADRE andHA-specific T cells (FIG. 23A), and then cultured with 10 uu/ml of PADREfor ten days. As shown in FIG. 23B, PADRE-specific T cells were expandedto 8.1%, while the number of cells specific for the control HA peptideactually decreased from 1.0 to 0.3% (FIG. 23D). Interaction betweenantigen-specific T cells and artificial APC was, as expected, dependingon availability of TCR and HLA/peptide molecules. Our experiment furthershowed that binding was inhibited by addition of anti-HLA antibody priorto incubation of artificial APCs with the T cells. Moreover, both totalcell number and MFI were reduced by 62% when compared with panel 23Bupon competition between biotynilated and non-biotynilated PADREpeptides. This control further supports the specificity of theinteractions between the various components of the system, and rules outthe possibility of steric hindrance from the biotin at the n-terminal ofthe peptide in interfering with the necessary molecular interactions.

T Cell Capture by Artificial APCs is More Sensitive than Measurement ofCytokine Production to Assess the Number of Antigen Specific T Cells ina Culture

Measurement of production of cytokines in response to an antigen isoften used to estimate the number of antigen specific T cells. Tocompare sensitivity of measuring cytokine production against T cellcapture by artificial APCs, we harvested culture supernatants fromPADRE-stimulated cultures, at the fourth and sixteenth day of cultureand measured concentrations of IL-2 and IFN-γ by sandwich ELISA. Asshown in FIG. 24, the differences in IL-2 and IFN-γ production betweenthe two time points were not as evident as the increase inPADRE-specific T cells, as measured by our T cell capture method (FIGS.23A-F). Hence, from the comparisons of the T cell capture usingartificial APCs with antigen induced T cell proliferation (FIGS. 25 and26) and cytokine production (FIG. 24) it appears evident that T cellcapture using artificial APCs is a tool for sensitive measurement ofantigen-specific responses. The advantage of identifying specific Tcells by T cell capture is also important for the fact that T cells maynot directly participate in antigenic responses while retaining antigenspecificity. This phenomenon may also provide an important tool for theunderstanding of the regulation of T cell responses to antigens.

Methods Short Term T Cell Line

Peripheral Blood Mononuclear Cells (PBMC) from healthy donors werestimulated in vitro with the PADRE peptide. PBMC were isolated using aFicoll-Isopaque gradient and stimulated in vitro with 10 ul/ml PADREpeptide in a 24 well tissue culture plate (Costar, Cambridge, Mass.) at4×10⁶ cells per well. The cells were cultured in RPMI containing 15% ABserum, at 37° C. and 5% CO₂. At days 4 and 7, media were replaced withfresh medium containing recombinant human Interleukin-2 (IL-2) at afinal concentration of 10 ng/ml. Culture supernatants taken at day 4were analyzed for production of IL-2 and Interferon-y by ELISA. At day10 viable cells were harvested and analyzed in a T cell capture asdescribed below.

Animals

Balb/C mice were obtained from Harlan. Mice were 4-6 weeks old at thebeginning of each experiment.

Immunizations

Balb/C mice were immunized with 100 mg of OVA³²³ ³³⁹ in CompleteFreundis Adjuvant (CFA) subcutaneous, followed by immunizations with 100mg of OVA³²³⁻³³⁹ in Incomplete Freundis Adjuvant (IFA) at days 7, 14,and 21 after the initial immunization. A subgroup of mice was sacrificed5 days after each immunization.

Preparation of MHC

Balb/C MHC class II molecules I-Ad and I-Ed were purified from thelysate of B cell lymphoma A20.11 by immunoanaffinity chromatographyusing anti-IAd monoclonal antibody MKD6 (Pierce) and anti-IEk monoclonalantibody 14-4-4S, essentially as previously described.

Human MHC Class II DRB1*0401 and MHC Class I molecules were purifiedfrom the lysate of the Epstein Barr virus transformed B cell lines.Affinity purified MHC molecules were solubilized in a TRIS buffercontaining 50 mM diethylamine and 0.2% n-acetyl-octyl-glucopyranoside(Calbiochem, San Diego). Conditions tested: pH5 and 7, RT and 37° C.,16-24-40 hr peptide loading.

Competition/Inhibition of Non-biotinylated PADRE Peptide on Binding toHLA DR4*0401

A modified ELSA using soluble HLA-DR 0401 assessed thecompetition/inhibition of biotinylated and non-biotinylated PADREpeptide. A ten-fold molar excess of biotinylated PADRE peptide wasincubated with affinity purified DR4*0401 at pH 7.0 for 16 hrs in roomtemperature. In addition, a 250 fold molar excess of non-biotinylatedPADRE was added. After 16 hours the complexes were transferred to a96-well flat bottom ELISA plate (Costar) coated with an anti-DR captureantibody. The excess of unbound peptides and complexes were removedtrough extensive washing with buffer. A 1:20,000 solution ofNeutravidin-HRP was then added to the wells and incubated for 40 minutesin room temperature. After washing, a TMB developing solution was addedand developed for approximately 15 minutes. IM H3PO4 was used as a stopsolution. The optical density was read at 450-650 nm using a micro-platereader. Delta Soft program was used to analyze the data.

Preparation of Cells

Cells were washed twice in staining buffer (PBS with 2% FCS and 0.05%sodium azide) and then incubated at 4° C. for 20 minutes in blockingbuffer (staining buffer with 10% FCS, for mouse cells FcBlock(Pharmingen) is used). The cells were stained for the surface markersand isotype controls for 20 minutes at 4° C., and washed twice andresuspended in staining buffer. The following monoclonal antibodies wereused: PE-, CY- and FITC-labeled anti-mouse and anti-human CD3 and CD4.

T Cell Capture

The cells were incubated with the MHC-peptide liposomes for 20 minutesat room temperature. Before acquisition on the FACScan, cells werewashed twice (5 minutes, 500 g) and resuspended in staining buffer.Partial inhibition of the interaction of cells to the liposomes wasachieved with monoclonal antibodies against T cell receptor or MHC. Forinhibition through partial blocking of the T cell receptor, cells wereincubated for 20 minutes with 50 ul/ml unlabeled anti-TCR antibody(Pharmingen), prior to the incubation with the liposomes. For inhibitionthrough partial blocking of MHC, the liposomes were incubated withunlabeled anti-MHC at a molar ratio of 1:1 to incorporated MHC. Forcompetition/inhibition with non-biotinylated peptide, non-biotinylatedpeptide was added together with the biotinylated peptide, and at thesame molar ratio to the MHC.

EXAMPLE 17 Characterization of Artificial APCs

Mean particle sizes of the MHC-containing liposomes were determinedthrough dynamic light scattering analysis with a Malvern 4700 system,using a 25 mW Ne—He laser and the automeasure version 3.2 software(Malvern, Ltd.). For refractive index and viscosity the values for purewater were used. The particle size distribution was reflected in thepolydispersity index (p.i.: 0, which ranges from 0.0 for a monodisperseto 1.0 for a polydisperse dispersion). Besides DLS analysis, liposomeswere visualized through flow cytometric analysis as described beforeherein. Briefly, FITC-labeled liposomes were gated according to theirFL1 fluorescence by placing a threshold at the FL1 channel, while forforward and side scattering the most sensitive settings were selected.The size of the FITC-liposomes was compared with the FSC of 2 types ofFITC-labeled calibration beads ranging in sizes from 40 to 60 nm andfrom 700 to 900 nm (Pharmingen). The results are shown in FIG. 20wherein the shaded area shows that the artificial APCs size peak is inthe range of 0.05 uM.

For qualitative analysis of the MHC class II incorporation efficiency asexplained in Example 16, MHC class-II liposomes were incubated withPE-labelled anti-MHC class II mAbs (anti-DR4 (Parmogen); anti-I-Ad(clone 0X-6, Harlan)) or PE-labelled isotype controls and analyzed forPE-staining on the FACS calibur. MHC incorporation was quantitativelyanalyzed by a Petterson modification of the Lowry protein assay.Briefly, a standard curve (0-1-2-4-6-8-12 ug of MHC) was made with thesame detergent solubilized MHC batch as used for the preparation of theartificial APC. A reliable range for protein determination was between2-12 mg of MHC. All samples were analyzed in duplicate and linearregression was performed on the standard curve to evaluate the amount ofprotein incorporated in the liposome preparation. To calculate thenumber of MHC molecules per liposome, the amount of MHC, as determinedby the Petterson modified Lowry method, and the size of the liposomes,as determined by DLS, were used to convert the amount of incorporatedMHC into the number of MHC per liposome, assuming that a 100 nm liposomecontains approximately 80,000 phospholipid molecules/vesicle, andassuming that the average area per phospholipid molecule is 75 uM².

For qualitative analysis of peptide loaded MHC class II molecules, MHCmolecules were preloaded with biotinylated-peptide using the optimizedpeptide-MHC loading conditions. After MHC incorporation, the artificialAPC were incubated with Streptavidin-CY (Pharmingen), and analyzed forFL3 staining using the FACS calibur. The quantitative analysis ofoccupancy of the number of MHC molecules with the desired peptide wasperformed in 2 different ways, namely, soluble ELISA and by the analysisof unbound biotinylated peptide after SDS-PAGE and blotting of MHCmolecules incubated with peptide.

In addition to the MHC samples incubated with a dose range ofbiotinylated peptide, the same dose range of biotinylated peptide wasincubated under exactly the same experimental conditions without theaddition of MHC. After 24 hrs., two gels were run under identicalconditions in one minigel-system (Biorad). One gel was loaded with theMHC-peptide samples, while the other gel was loaded with the peptidesamples. The fronts of the gels were scanned by using the MolecularAnalyst software (Maxsott), and a standard curve was made by linearregression of the peptide loaded gel. After front analysis of theMHC-peptide gel the decrease of the peptide signal was quantified bycomparison with the standard curve and consequently, the amount of MHCbound peptide could be determined. See FIG. 19.

Methods Peptide Loading of MHC Class II Molecules

To determine optimal loading conditions for MHC class II molecules a MHCclass II binding assay was performed, essentially as described beforewith minor modifications. Briefly, detergent solubilized MHC class IImolecules (0.5-1 uM) were incubated with a 50-250-fold molar excess ofbiotinylated peptide for the designated time, pH and temperature withoutthe addition of protease inhibitors. The following conditions weretested; pH 5 and pH 7; Room Temperature and 37° C.;16, 24 and 40 hoursof peptide loading; 0.05-, 1-, 10-, 100-fold molar excess of peptide.For DRB1*0401 0.5 uM, for I-Ad 3 uM of MHC was used in binding assays.

MHC-peptide complexes were analyzed via a non-reducingSDS-polyacrylamide gel electrophoresis (SDS-PAGE). Following WesternBlotting (Highland-ECL, Amersham), the biotinylated peptides werevisualized on preflashed films (Hyperfilm, Amersham) through enhancedchemiluminescence (Western blot ECL kit, Amersham). Optimal bindingconditions were established by evaluation of the density of the spots atthe position of the MHC class II dimer-peptide complexes. The presenceof the MHC Class II dimer under the tested conditions was checked with anon-reducing SDS-PAGE followed by silver staining (Biorad). (see FIGS.19A-D)

Preparation of Liposomes

Liposomes were prepared as follows. Stock solutions of egg PhosphatidylCholine (Sigma) and Cholesterol (Sigma) were combined in a glass tube ata molar ratio of 7:2.N-(fluorescein-5-thiocarbamoyl)-1,2-d-ihexadecanoyl-sn-glyvero-3-phosphoethanolamine,triethylammonium salt (fluorescein-DHPE, Molecular ProbesEugene, Oreg.)was added at a final concentration of 1 mol % of Phosphatidyl Choline.The solvent was evaporated under an Argon stream for 30 minutes and thelipids were dissolved at a final concentration of 10 mg/ml in a buffercontaining 140 mM NaCl, 10 mM TrisHCl (at pH 8) and 0.05% deoxycholate(Buffer A). The solution was sonicated until clear, and aliquoted in 500ul Eppendorf tubes and stored at minus 200° C.

Incorporation of MHC-peptide in Liposomes

After preincubation of biotinylated peptides and MHC at conditions basedon in vitro MHC binding, MHC-peptide complexes were added to the lipidsin Buffer A at a ratio (w/w) of MHC: liposomes of 1:7 (DR4) or 1:10 (IAdand IEd). Additionally, for each T cell capture sample a ratio of 1 ulMHC per 6000 cells was used.

The lipids and MHC-peptide complexes were dialyzed at 4° C. against PBSin a 10K Slide A Lyzer (Pierce) for 48 hours with two buffer changes.The formed liposomes were then recovered from the Slide A lyzers andincubated with streptavidin-CY for 20 minutes before adding theliposomes to the cells. For MHC staining, liposomes were stained with aPE-labeled anti-MHC antibody at the same time.

Where other molecules of interest are to be incorporated into theartificial APC (i.e., accessory, adhesion, co-stimulatory, cellmodulation, irrelevant, GM-1 ganglioside molecules, and cholesterolmolecules), such molecules can be added simultaneously with theincorporation of the MHC:antigen complexes.

Quantification of HLA-DR and PADRE Peptide Bound to Liposomes

A modified ELISA was used to assess the amount of HLA-DR bound to theliposomes. Briefly, liposomes containing HLA DR4*0401 and biotinylatedPADRE peptide were formed as described above. The resulting liposomeswere counted and sorted by FACS on (FITC) fluorescence and forwardscatter. The sorted samples were then divided into two lots for HLA andpeptide quantification by ELISA.

DR Quantification

The sorted liposome sample was initially incubated with 10× M excess ofneutra-avidin (Pierce) to cap the biotin on the peptide. This wasfollowed by an extensive dialysis with a 300 k MWCO membrane (Pierce) toremove excess unbound neutra-avidin. The sample was then incubated withbiotinylated mouse anti-DR (Parmagen) at 1:1 molar ratio, which wasagain followed by extensive dialysis. The tagged complex was incubatedwith neutra-avidin-HRP at 1:20,000 and the excess dialyzed as before. Aknown amount of soluble DR pre-incubated with non-biotinylated PADREpeptide (standard) was incubated with the same antibody used fordetecting the DR in the liposomes. Multiple epitope recognition by theantibody assured retention of the complex during dialysis steps. Boththe sorted sample and the standard were then developed with TMB-HRPsubstrate and read at 450-650 nm.

PADRE Peptide Quantification

The sorted liposome sample was incubated with non-biotinylated anti-DR(Pharmagen) at a 1:1 molar ratio. The sample was treated and developedin the same manner as for DR quantification described above. The peptidewas quantified based on its biotin tag. Analysis of the data wasadjusted to background and to appropriate negative controls.

EXAMPLE 18 Methods for Orienting Molecules of Interest in as ArtificialAPC

We have shown that incorporation of molecules of interest into theartificial APC liposome matrix yields insertion of such molecules in anorientation wherein the active center of the molecules face outward onthe APC at a rate of about 50%. We have found that proper orientationcan be dramatically increased to over 90% by applying a mechanism todirect the insertion of the molecules. This mechanism uses GM-1ganglioside, which is a pentasaccharide that acts like a transmembraneprotein and has an affinity for binding the β subunit for cholera toxin.When the GM-1 is associated with the liposome membrane of the APC, itcan be used to bind cholera toxin which in turn can be attached to themolecule of interest. By attaching the cholera toxin to a point distalto the active site of the molecule of interest, we can direct theorientation of the molecule of interest such that the active site willbe placed in the artificial APC in an orientation favorable tointeraction with T cells and various molecules. In a preferredembodiment, proper orientation is obtained using a recombinant fusionbetween the molecule of interest and the β subunit of cholera toxin theconstruction of which will be well understood by those skilled in theart of making recombinant fusion proteins. The β subunit can also beconnected to a molecule of interest using a linker.

Methods GM-1 Ganzliosie Pentasaccharide Anchors

The structure of GM-1 gangliosides has been elucidated and shown to bepentasaccharide. The molecule is branched having terminal sugars,galactose and sialic acid (n-acetyl neuraminic acid), which exhibitsubstantial specific binding interactions with the Psubunit of choleratoxin. A smaller contribution to binding is derived from the N-acetylgalactose residue of the molecule. This binding interaction is mediatedthrough hydrogen bonding. Each of the five identical binding sites areprimarily within a single monomer of the B-pentamer. GM-1 gangliosidemay be purchased from commercial sources (Sigma #G7641).

Cholera β Subunit (CTB)

Cholera toxin (mw=84 kda) is comprised of two subunits; a (mw=27 kda)and β(mw=1 1.6 kda). The amino acid sequence has been determined atabout 11,604 da. The primary structure of the β subunit, which isresponsible for binding of the toxin to the cell receptor gangliosideGM1, has been determined. Cholera toxin's ability to bind well to suchtransmembrane structures makes it very attractive anchor for membraneproteins (e.g. molecules of interest as disclosed herein). Since the βsubunit is primarily involve in the binding to the GM1 gangliosidepentasaccharide, the α subunits are not necessary. The use therefore ofonly the β subunit makes issues respecting toxicity less important withrespect to use of the toxin protein subunits in therapeutic applicationssuch as drug delivery or transport and manipulation of T cells ex vivo.

The receptor binding site on the CTB is found to lie primarily within asingle β-subunit, with a solvent-mediated hydrogen bond involving thetwo terminal sugars of GM1 (galactose and sialic acid). The binding ofGM1 to cholera toxin thus resembles a 2-fingered grip. Cholera toxinβ-subunit may be purchased from commercial sources (Sigma #C9903 orC7771) or synthesized by recombinant methodology.

Linkers for Attaching Cholera Toxin to Molecules of Interest

Linkers may be used to attach the cholera toxin subunit to the moleculeof interest. In a preferred embodiment, a linker such asN-succinimidyl[3-(2-pyridyl) dithio) propionate (SPDP, Sigma Prod. No.P3415). NHS-esters such as this yield stable products upon reaction withprimary or secondary amines. Coupling is relatively efficient atphysiological pH. Accessible -amine groups present on the N-termini ofproteins react with NHS-esters and form amides. In this regard, reactionwith side chains of amino acids can also occur. A covalent amide bond isformed when the NHS-ester cross-linking agent reacts with primaryamines, releasing N-hydroxysuccinimide.

In another preferred embodiment, pyridyl disulfides can be used to reactwith sulfhydryl goups to form a disulfide bond. Pyridine-2-thione isreleased as a by-product of this reaction. These reagents can be used ascross-linkers and to introduce sulfhydryl groups into proteins.Conjugates prepared using these reagents are cleavable.Pyrimidine-2-thione groups are released upon reaction with free —SHs andthe concentration can be determined by measuring the absorbance at 343nm (Molar extinction coefficient=8.08×10⁻³ M⁻¹ cm⁻¹).

Results

The GM-1 ganglioside molecule acts like a transmembrane protein byanchoring itself in the lipid bilayer. It can be associated with the“raft” or freely mobile molecules of interest in the lipid membrane.GM-1 ganglioside has been used in studying movement and interactions ofco-stimulatory molecules through cross-linking the molecule withflourescinated-tagged cholera toxin. This cross linking can be carriedout in several ways as shown in FIG. 27. For example, in FIG. 27A, asynthetic gene encoding a molecule of interest is cloned into acommercial vector such as a generic expressions vector (for example, DESExpression Vector, Invitrogen) and expressed. The recombinant productcan be purified and linked to GM-1 protein that is in an artificial APC.In a similar fashion (FIG. 27B) the molecule of interest can be clonedinto an expression vector as a fusion protein with cholera toxin βsubunit. The fusion product can be purified and mixed with an artificialAPC containing GM-1 ganglioside where the cholera moiety will bind tothe GM-1 ganglioside. Additionally, as shown in FIG. 27C, the choleratoxin (whether natural or recombinant) can be attached to a linker, suchas N-succinimidyl [3-(2-pyridyl) dithio]propionate, either to a completeβ subunit molecule or during synthesis of a recombinant toxin molecule,the product of which can be then mixed with a GM-1 gangliosidecontaining artficial APC.

We have found that cross-linkers are useful as “flexible hinges” whereprotein molecules can be covalently linked to allow for intercellularinteraction with transmembrane proteins (e.g., B7-CD28, ICAM-1-LFA-1,MHCs, TCRs, etc.). Once the GM-1 ganglioside protein is incorporatedinto the liposome of the aAPC, cholera toxin-cojugated surface proteinscan then be cross-linked. The system containing properly orientedmolecules of interest can then be tested through ELISA, WESTERN, and byFACS analysis.

EXAMPLE 19 High Density Expression on aAPC of Molecular Rafts ComprisingGM and Molecules Active in T-Cell/aAPC Interactions

Stable interactions between aAPCs and T-cells depends on severalfactors. These include the absolute affinity between TCRs and theirrespective ligands, as well as the relative density of moleculesavailable for interaction at the binding site. Under normal physiologicconditions, ligand density is achieved in part by migration of therelevant molecules in the cell membrane toward the site of initial Tcell-APC interaction, i.e., capping as described earlier, the outcome ofwhich is stable cell-cell binding. Clustering ensures specificinteraction between T-cells and APCs.

The molecular interaction between the clustered transmembrane moleculestaking part in T-cell/APC recognition has been termed the “immunesynapse”. The outcome of forming the immune synapse is an antigenspecific response by the T-cell mediated by signaling through the TCR.

Several factors contribute to significant quantitative and qualitativedifferences in the response provided by the T-cells. These factorsinclude TCR affinity for the MHC/peptide complex and the number ofligands available for interaction. The threshold of response is achievedwhen enough ligands have migrated to the initial interaction site on thecell surface. Prior to the current invention, known conceptionsconcerning liposome formation and uses thereof precluded the ability toachieve high threshold immune responses. However, as shown by theresults obtained for responses induced by aAPC of the current invention,use of GM-1 based raft structures in liposomes of the particular designof the current invention provide a markedly and unexpectedly superiorresponse result.

The current invention as shown in this example recognizes thatmodulation of antigen specific T cell response is affected and can bedirected by the ability to control the affinity and molarity of themolecules involved in T-cell binding and activation. Our uniqueconstruction of GM-1 containing AAPC allow for control of the contentand relative ratios of molecules used on the AAPC for interaction withT-cells. We are further able to produce the aAPC with relative highdensities of such molecules. Additionally, we provide examples of theeffectiveness of such aAPC in binding and activation of T-cells. By“high density” is mean that the aAPC of the current invention possesssubstantially elevated numbers of immunologically active molecules onfreely mobile rafts per each aAPC sufficient to support at least bindingbetween aAPC and a T cell and/or immunmodulation of a T cell.

Artificial APCs are constructed with free floating rafts in the lipidmembrane of the aAPC wherein the rafts contain high density of moleculesthereby providing hundreds of molecules that are available for bindingby the T-cell which collectively are capable of comigration, as in thecapping process, mimicing the physiologic interaction between T-cell andnatural APC. Hence, both high absolute numbers and relativeconcentrations of interacting molecules are achieved on a uniquelydesigned freely migratable raft using the aAPC of the present invention.

aAPC Raft Composition

In this embodiment, rafts, also referred to as “micromembrane domains”are formed in the bilayer lipid membranes of aAPCs. By “rafts” or“micromembrane domains”, is meant the free-floating molecular complexwhich interacts with the transmembrane molecules of the T cell that inpart form the immune synapse as is understood by one of skill in therelevant art. In a preferred embodiment, the rafts comprise multiples ofGM-1 bound to molecules that interact with the T cell. In a preferredembodiment, each unit of the GM-1 for example is bound to a choleratoxin β subunit which is in turn fused with a molecule of interest suchas a molecule that is active in some manner with the T cell/aAPC bindingor T cell modulation. Generally, the T cell interacting proteins may befusion proteins comprising at least three components. These componentsare (1) an immunologically active component for binding or otherwiseinteracting with T-cells, (2) a variety of linker components that cancomprise short peptides or functional peptides (such as an amino acidsequences capable of binding other peptide sequences), and (3) an anchorcomponent comprising the cholera toxin subunit β itself. By“immunologically active molecule/protein”, is meant molecules that areused to bind to T cells and/or participate in the immunemodulation of Tcells, or that can be used to detect location of molecules either on theT cell surface or surface of the aAPC.

Fusion protein constructs expressed in appropriate host expressionsystems well known to those of skill in the art may be isolated for usein the rafts. In forming the rafts, each of the cholera subunits makingup the carboxyterminal portion of the fusion proteins becomes bound byone GM-1. Thus, for every raft, due to natural aggregation of GM-1within the lipid bilayer (typically to form a pentameric structure), atleast five immunologically active molecules can be associated with eachraft present on the aAPC. As shown below, the immunologically activemolecules can be mixed and matched as desired to create a specific ratioand density of molecules for binding to and modulation of the T cells.FIG. 28, shows the high density expression concept described above.

In one embodiment, the fusion proteins can be designed as cassettes inexpression plasmids comprising the various components (i.e., choleratoxin, linker, immunologically active molecule). Generally, the choleratoxin anchor component can be constructed as a cDNA cassette in anexpression plasmid comprising sequence encoding, from 5′ to 3′, a shortlinker sequence followed by the toxin sequence. The linker sequencecodes for appropriate restriction endonuclease sites and a stretch ofamino acids to provide spacing between the toxin subunit and theimmunologically active component that is desired for attachment to thecassette. Alternatively, both a linker sequence and an additionalnucleic acid sequence coding for an amino acid sequence having a bindingfunction for binding additional molecules important in T cell bindingand modulation can be constructed into the cassette. The cassette canthen be manipulated, through recombinant technology well known to thoseof skill in the art, such that the linker sequence is fused to asequence coding for an immunologically active protein of interest. Onceso constructed, the expression plasmid may be processed for ultimateproduction and isolation of the resulting fusion protein. The fusionprotein may then be used in forming high density rafts for insertioninto the aAPC.

Generally, the immunologically active portion of the fusion proteins canbe any sequence recognized by the T-cell and/or important to T cellbinding and/or immumodulation, or detection of the binding process.Examples of such proteins are, without limitation:

-   -   (a) MHC molecules;    -   (b) Antibody molecules that interact directly with the TCR;    -   (c) Co-stimulatory molecules including, but not limited to,        B7-1, B7-2, OX40, chemokine receptors, CD30, CD5, CD9, CD2, and        CD 40, and many other receptors, ligands, and antibodies thereto        as would be understood as important to one of skill in the art;    -   (d) Antibodies directed against CD4 and CD8 receptors, tissue        specific receptors, syalic acid;    -   (e) Cytokines such as interferon and IL-4;    -   (f) Accessory molecules as delineated throughout this disclosure        including, but not limited to, LFA-1, CD11a/18, CD54(ICAM-1),        CD106(VCAM), and CD49d/29(VLA-4).    -   (g) Antibodies to accessory molecules;    -   (h) Antibodies against CD28 and CTLA4;    -   (i) Adhesion molecules including, but not limited to, ICAM-1,        ICAM-2, GlyCAM-1, CD34, anti-LFA-1, anti-CD44, anti-beta 7        antibodies, and chemokine receptors such as CXR4, and CCR5;    -   (j) T cell modulatory molecules including, but not limited to,        CD72, CD22, and CD58; and    -   (k) Irrelevant molecules for binding the aAPC to solid supports        or for use as a label.

Immunologically active molecules such as those provided above whenpresented in the high density component rafts of the invention aid inthe binding and/or modulation of the T-cells which come into contactwith the aAPCs.

For example, FIG. 29 shows a fusion construct wherein a nucleic acidsequence encoding the B7-1 molecule has been attached to the cholera Psubunit nucleic acid sequence through a linking sequence (underlined).

Specifically, the B7-1 portion makes up the immunologically activeportion which comprises the N-terminal region of the fusion proteinwhile the cholera subunit (fused through the linker to its N-terminalamino acid) makes up the C-terminal portion.

In another example, shown in FIG. 30, a very similar construct can becreated for the B7-2 molecule.

As will be understood by those skilled in the art, appropriate makeup ofthe fusion proteins is amenable to computer analysis for determiningsequences for fusion constructs so as to predict and avoid sterichindrance and undesirable tertiary interactions of the amino acidmoieties on the immunologic portion with that of the cholera toxinanchor portion.

In yet another embodiment, in addition to the cholera toxin anchorportion, the immunologic portion, and linker portion, the fusionconstruct may also be designed to incorporate a protein binding peptidesequence. Such sequence can be, without limitation, a peptide such as aleucine zipper sequence as shown below: Leucine Zipper A:SAQLEWELQALEKENAQLEWELQALEKELAQ (Seq. Id. No. 19) Leucine Zipper B:AQLKKKLQALKKKNAQLKQKLQALKKKLAQ (Seq. Id. No. 20)

As shown in FIGS. 31 and 32, immunologically active proteins such as theHLA FRA1 and DRB1*0401 sequences comprising α and β domains can beengineered into a fusion protein constructs. In FIG. 31, the α domain(comprising α1 to α2) forms the N terminal portion of the fusion whichis then linked by a short amino acid sequence to Leucine zipper Afollowed by a second linker sequence which is followed in turn by thecholera toxin β subunit making up the carboxy-terminal portion of thefusion.

FIG. 32 shows the construction of the HLA DRB1 *0401 β domain(comprising β1 to β2) fused to a short amino acid linker, followed byLeucine zipper B, followed in turn by a second linker, which is followedyet again by a biotag peptide sequence. As described earlier, the biotagmoiety comprises an irrelevant molecule in that it does not participatein binding between T cells and the aAPC or in immunmodulation but doesprovide a means for visualization of the T cell-aAPC binding process.

In the embodiment comprising DRA and DRB fusion proteins, we demonstratethe ability to create highly complex rafts wherein one fusion proteinconstruct possessing a cholera anchor segment that is bound to a GM-1moiety of the raft, is able to carry with it another fusion protein thathas no cholera anchor component. Specifically, as shown in FIG. 33, theA and B Leucine zippers bind to one another allowing the rafts to carryfusion proteins comprising both α and β domains. This allows forinsertion of complex molecular structures into the raft without using acholera toxin anchor for at least one molecule making up the complexstructure. Further, the use of binding sequences (such as the leucinezipper) allows for the proper orientation and tertiary structure betweenmolecules that must form a specific three dimensional structure (as isthe case with the HLA-DRB1) to achieve its immunologic usefulness.

From the above examples of fusion protein constructs, it can readily beunderstood that aAPC can be generated containing high density raftscomprising any combination and/or ratio of immunologically activemolecules. The ability to control raft structure contrasts substantiallywith current methods for ‘detection’ of antigen specific T-cells. Forexample, existing systems used in binding assays rely on absoluteaffinity between the T cell and the molecular complexes. Moreover,current binding assays are limited in the nature of the bindinginteraction between T cells and binding complexes comprisingimmunologically active molecules because they do not allow thephysiologic phenomenon of capping. Such systems must rely only on highaffinity binding which limits the use of such complexes to the detectionof binding but not manipulation of T cell modulation. Additionally, suchlimited methodologies may not have the capacity to detect “low affinity”binding interactions, which can be important to T celleducation/stimulation such as the induction of autoimmunity.

The current invention sharply contrasts with less capable methods by,for example, allowing active clustering of TCR-MHC molecules at theimmune synapse, which clustering occurs after the initial interactionbetween aAPC and T cells. The capability for this multivalent system forstrong binding aids low affinity interactions thereby providing for bothsimultaneous stimulation and modulation responses of antigen specific Tcells even in cases where the molecules comprising the immune synapseinteract with low affinity binding.

Besides the fusion protein concept described above, we have alsoengineered liposomes containing high density rafts by bindingneutravidin (also known as tetravidin) to the cholera toxin through abiotin moiety (FIG. 34). Since neutravidin has four binding centerscapable of binding four biotin molecules, each cholera anchor molecule(bound to a GM-1 molecule) can provide for binding three immunologicallyactive molecules through the neutravidin moiety. Specifically, as shownin FIG. 34, biotinylated cholera toxin is bound to GM-1 ganglioside. Thebiotin of the cholera toxin is bound to neutravidin that is in turnbound to any variety of biotinylated immunologically active molecules ofinterest. Hence, each GM-1 molecule of the rafts has three valencesavailable for interaction with T cells. Since the GM-1 moleculesaggregate to form generally pentameric rafts, it is possible for atleast 15 immunologically active molecules and as many as 30immunologically acitive molecules to be present on any pentamericGM-1-based raft. This raft construct method ultimately results in thepossibility to provide several thousand immunologically active moleculeson several hundred high density rafts on each aAPC. FIG. 34 depicts thestructure of a hypothetical individual GM-1 raft in the aAPC membrane.

To show raft density on the aAPCs, FIG. 35 provides FACS analysis ofraft densities wherein various raft components are added using thetetravidin construct method. The data shows binding of biotinylatedcholera toxin to GM-1 in the aAPC. In this experiment, the cholera toxinis conjugated with FITC to allow visualization of the binding of thecholera toxin to the aAPC. FIG. 35A depicts the liposome containing onlyGM-1 molecules. FIG. 35B depicts binding of the cholera toxin to theGM-1 molecule at substantially high levels. These levels, as shown inFIG. 36 correspond to a binding efficiency approaching 100% saturationof all GM-1 molecules on the aAPC. Specifically, the aAPC, which werecreated using 230 ug of total lipid and 10 ug of the GM-1 (i.e.,approximately 6374 pico moles), were incubated with various amounts ofFITC conjugated cholera toxin for 20 minutes, washed, and read fordensity. Saturation of the GM-1 with cholera toxin begins to occur atlevels in the area of 5,000 pico moles of cholera toxin showing thateach aAPC can contain hundreds of GM-1/cholera toxin raft formingmicrodomains expressing even more hundreds of immunologically activemolecules available for binding (i.e., high density rafts).

To show the efficiency of aAPC of the current invention and method overconventional liposome art, high density rafts incorporating HLA, HA(antigen peptide) and anti CD28 were tested for binding with CD4+T cellsfrom a healthy individual previously immunized with flu vaccine. Thepercent of CD 4+ cells bound by the aAPC was observed by FACS analysis.Visualization of binding was obtained by adding FITC conjugated choleratoxin to the aAPC prior to incubation with PBMC.

As shown in FIG. 37, the GM based raft containing aAPC exhibited asignificant increase of bound CD4+ cells over that of non-raftliposomes. Specifically, GM-1 based raft aAPC containing HLA/HA and acostimulous anti CD28 molecule was presented to CD4+ cells and comparedto liposomes prepared only with HLA and HA. In the non-raft liposomes,the HLA and HA are randomly distributed throughout the liposome lipidbilayer. These results show that the aAPC of the current invention aresubstantially superior in binding T cells over the binding capability ofprior liposome construct concepts.

Additionally, as shown in FIGS. 38A and B, binding to T cells by aAPCcontaining GM-1 rafts with HLA and HA alone is compared to binding ofcells by non-raft liposomes containing only randomly distributed HLA andHA. Here, the costimulatory effect of anti CD28 is not present. Thus,the results show that the raft based aAPC provided at least a 100%greater efficiency of binding to T cells. Therefore, the aAPC of thecurrent invention is proven substantially superior to prior liposomemethods and further indicates that the current invention's employment ofGM-1 based rafts bearing immunologically active molecules providesunexpected and significantly improved binding to T cells over that ofprior liposome construct methodologies.

EXAMPLE 20 T Cell Modulation by Aapc

One of the most important and original advantages of the technologyherein disclosed is that it provides a tool to control exactly theaffinity and molarity of molecules on the aAPC surface at the level ofmicromebrane domains. This is an essential and, to date, unfulfilledrequisite for controlling T cell responses. Below are provided twoexamples of such control. In the first, the GM-1 based raft containingaAPC is compared with liposome constructs of previous methods, instimulation of T cells in a non-antigen specific fashion. In the secondexample, the capability of the aAPCs of the present invention tomodulate ex vivo T antigen specific T cell responses is demonstrated.

To compare our raft technology with prior art in stimulation of T cellin a non antigen specific fashion, CD4+ cells were sorted from PBMC(using PBMC from the same donor as described above) followed byincubation for 72 hours with either AAPC containing anti CD3 and antiCD28 antibodies on GM-1 based rafts, or with anti CD3 and anti CD28 thathad been bound to a planar surface of a tissue culture plate well asdisclosed in prior applications. The CD4+ cells were tested forexpression of IL-2, a cytokine associated with T cell proliferation andactivation. Analysis was performed by FACS.

As shown in FIGS. 39A and B, activation of CD4+ cells using GM-1 basedaAPC of the current invention provided significantly higher efficiencyin expression of IL-2 than that of the solid phase methodology. Theresults further indicate that efficiency of stimulation was strictlydependant not upon the engagement of the CD28 receptor by the anti CD28antibody but on the organization of the immunologically active moleculescomprising the high density raft constructs of the current inventionaAPCs.

In another experiment the ability to modulate T cells was tested bycomparing CD69 (FIG. 40) and IL-2 (FIG. 41) production in CD4+ sortedcells incubated with aAPC containing either CD3 or CD28, or both CD3 andCD28. When both CD3 and CD28 are present, IL-2 and CD69 production aresubstantially stimulated. This shows that the aAPC of the currentinvention are capable of providing an effective means for modulation ofT cell response in vitro.

To demonstrate the capability of our invention to modulate antigenspecific T cell responses, we incubated CD4+ cells from a flu-vaccinateddonor with aAPC containing high density micromembrane domains expressingHLA/HA complexes, with or without costimulatory molecules, and comparedCD69 expression and IL-2 production with prior art (i.e. liposomes inwhich HLA/HA peptide complexes were randomly distributed on thesurface). as shown in FIGS. 42 and 43 such responses can be specific innature. In FIG. 42 CD69 production by CD4+ cells is significantlygreater using aAPC of the current invention wherein the aAPC contain HLAand HA and anti CD28 as compared to non-GM-1 based liposomes. FIG. 43provides similar results for IL-2 expression. In both cases, specificstimulation using the aAPC of the current invention is approaching 3fold increases in stimulation over that of non raft liposomes.

These results therefore demonstrate the superiority of our technologyover prior approaches. aAPC engineered to contain micromembrane domainscan modulate in a specific fashion antigen specific T cells responses,thus providing an important tool for ex vivo therapeutic, diagnostic andresearch applications.

Modifications and other embodiments of the invention will be apparent tothose skilled in the art to which this invention relates having thebenefit of the foregoing teachings, descriptions, and associateddrawings. The present invention is therefore not to be limited to thespecific embodiments disclosed but is to include modifications and otherembodiments which are within the scope of the appended claims. Allreferences are herein incorporated by reference.

Sequence CWU 1

24 1 17 PRT Artificial Sequence Synthesized peptide derived from thirdhyper V region of IE molecule Mus musculus Ala Ser Phe Glu Ala Gln GlyAla Leu Ala Asn Ile Ala Val Asp Lys 1 5 10 15 Ala 2 15 PRT ArtificialSequence Synthesized peptide derived from bole I protein of Epstein Barrvirus 2 Thr Arg Asp Asp Ala Glu Tyr Leu Leu Gly Arg Glu Ser Val Leu 1 510 15 3 16 PRT Artificial Sequence Synthesized peptide derived from thehemophilus influenza virus 3 Thr Ser Phe Pro Met Arg Gly Asp Leu Ala LysArg Glu Pro Asp Lys 1 5 10 15 4 36 PRT Artificial Sequence Synthesizedpeptide derived from the TCR receptor gene of Mus musculus 4 Leu His IleSer Ala Val Asp Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ser 1 5 10 15Ser Gln Glu Phe Phe Ser Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr 20 25 30Arg Leu 35 5 9 PRT Artificial Sequence Synthesized peptide derived fromthe influenza virus 5 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 6 9 PRTArtificial Sequence Synthesized peptide derived from the influenza virus6 Val Lys Leu Gly Glu Phe Tyr Asn Gln 1 5 7 11 PRT Artificial SequenceSynthesized peptide totally artificial 7 Lys Xaa Val Ala Ala Trp Thr LeuLys Ala Ala 1 5 10 8 13 PRT Artificial Sequence Synthesized peptidederived from the influenza virus 8 Pro Lys Tyr Val Lys Gln Asn Thr LeuLys Leu Ala Thr 1 5 10 9 17 PRT Artificial Sequence Synthesized peptidederived from the ovalbumin of Mus musculus 9 Ile Ser Gln Ala Val His AlaAla His Ala Glu Ile Asn Glu Ala Gly 1 5 10 15 Arg 10 15 PRT E. colidnaJpl heat shock protein 10 Gln Lys Arg Ala Ala Tyr Asp Gln Tyr Gly HisAla Ala Phe Glu 1 5 10 15 11 15 PRT Homo sapiens 11 Gln Lys Arg Ala AlaVal Asp Thr Tyr Cys Arg His Asn Tyr Gly 1 5 10 15 12 9 PRT Homo sapiens12 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 13 9 PRT Homo sapiens 13 ValLys Leu Gly Glu Phe Tyr Asn Gln 1 5 14 13 PRT Homo sapiens 14 Pro LysTyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr 1 5 10 15 313 PRT ArtificialSequence Fusion constructs with human and bacterial sequences 15 Met GlyHis Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr 1 5 10 15 LeuAsn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys 20 25 30 SerGly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 SerCys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 TyrTrp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85 90 95Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100 105110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 115120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg ArgIle 145 150 155 160 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His LeuSer Trp Leu 165 170 175 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr ThrVal Ser Gln Asp 180 185 190 Pro Glu Thr Glu Leu Tyr Ala Val Ser Glu PheGly Gly Ser Gly Gly 195 200 205 Ser Ala Thr Pro Gln Asn Ile Thr Asp LeuCys Ala Glu Tyr His Asn 210 215 220 Thr Gln Ile His Thr Leu Asn Asp LysIle Phe Ser Tyr Thr Glu Ser 225 230 235 240 Leu Ala Gly Lys Arg Glu MetAla Ile Ile Thr Phe Lys Asn Gly Ala 245 250 255 Thr Phe Gln Val Glu ValPro Gly Ser Gln His Ile Asp Ser Gln Lys 260 265 270 Lys Ala Ile Glu ArgMet Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr 275 280 285 Glu Ala Lys ValGlu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His 290 295 300 Ala Ile AlaAla Ile Ser Met Ala Asn 305 310 16 942 DNA Artificial Sequence Fusionconstructs with human and bacterial sequences 16 atgggccaca cacggaggcagggaacatca ccatccaagt gtccatacct caatttcttt 60 cagctcttgg tgctggctggtctttctcac ttctgttcag gtgttatcca cgtgaccaag 120 gaagtgaaag aagtggcaacgctgtcctgt ggtcacaatg tttctgttga agagctggca 180 caaactcgca tctactggcaaaaggagaag aaaatggtgc tgactatgat gtctggggac 240 atgaatatat ggcccgagtacaagaaccgg accatctttg atatcactaa taacctctcc 300 attgtgatcc tggctctgcgcccatctgac gagggcacat acgagtgtgt tgttctgaag 360 tatgaaaaag acgctttcaagcgggaacac ctggctgaag tgacgttatc agtcaaagct 420 gacttcccta cacctagtatatctgacttt gaaattccaa cttctaatat tagaaggata 480 atttgctcaa cctctggaggttttccagag cctcacctct cctggttgga aaatggagaa 540 gaattaaatg ccatcaacacaacagtttcc caagatcctg aaactgagct ctatgctgtt 600 agcgaattcg gcggctccggtggtagcgcc acacctcaaa atattactga tttgtgtgca 660 gaataccaca acacacaaatacatacgcta aatgataaga tattttcgta tacagaatct 720 ctagctggaa aaagagagatggctatcatt acttttaaga atggtgcaac ttttcaagta 780 gaagtaccag gtagtcaacatatagattca caaaaaaaag cgattgaaag gatgaaggat 840 accctgagga ttgcatatcttactgaagct aaagtcgaaa agttatgtgt atggaataat 900 aaaacgcctc atgcgattgccgcaattagt atggcaaatt aa 942 17 1056 DNA Artificial Sequence Fusionconstructs with human and bacterial sequences 17 atgggactga gtaacattctctttgtgatg gccttcctgc tctctggtgc tgctcctctg 60 aagattcaag cttatttcaatgagactgca gacctgccat gccaatttgc aaactctcaa 120 aaccaaagcc tgagtgagctagtagtattt tggcaggacc aggaaaactt ggttctgaat 180 gaggtatact taggcaaagagaaatttgac agtgttcatt ccaagtatat gggccgcaca 240 agttttgatt cggacagttggaccctgaga cttcacaatc ttcagatcaa ggacaagggc 300 ttgtatcaat gtatcatccatcacaaaaag cccacaggaa tgattcgcat ccaccagatg 360 aattctgaac tgtcagtgcttgctaacttc agtcaacctg aaatagtacc aatttctaat 420 ataacagaaa atgtgtacataaatttgacc tgctcatcta tacacggtta cccagaacct 480 aagaagatga gtgttttgctaagaaccaag aattcaacta tcgagtatga tggtattatg 540 cagaaatctc aagataatgtcacagaactg tacgacgttt ccatcagctt gtctgtttca 600 ttccctgatg ttacgagcaatatgaccatc ttctgtattc tggaaactga caagacgcgg 660 cttttatctt cacctttctctatagagctt gaggaccctc agcctccccc agaccacgaa 720 ttcggcggct ccggtggtagcgccacacct caaaatatta ctgatttgtg tgcagaatac 780 cacaacacac aaatacatacgctaaatgat aagatatttt cgtatacaga atctctagct 840 ggaaaaagag agatggctatcattactttt aagaatggtg caacttttca agtagaagta 900 ccaggtagtc aacatatagattcacaaaaa aaagcgattg aaaggatgaa ggataccctg 960 aggattgcat atcttactgaagctaaagtc gaaaagttat gtgtatggaa taataaaacg 1020 cctcatgcga ttgccgcaattagtatggca aattaa 1056 18 351 PRT Artificial Sequence Fusion constructswith human and bacterial sequences 18 Met Gly Leu Ser Asn Ile Leu PheVal Met Ala Phe Leu Leu Ser Gly 1 5 10 15 Ala Ala Pro Leu Lys Ile GlnAla Tyr Phe Asn Glu Thr Ala Asp Leu 20 25 30 Pro Cys Gln Phe Ala Asn SerGln Asn Gln Ser Leu Ser Glu Leu Val 35 40 45 Val Phe Trp Gln Asp Gln GluAsn Leu Val Leu Asn Glu Val Tyr Leu 50 55 60 Gly Lys Glu Lys Phe Asp SerVal His Ser Lys Tyr Met Gly Arg Thr 65 70 75 80 Ser Phe Asp Ser Asp SerTrp Thr Leu Arg Leu His Asn Leu Gln Ile 85 90 95 Lys Asp Lys Gly Leu TyrGln Cys Ile Ile His His Lys Lys Pro Thr 100 105 110 Gly Met Ile Arg IleHis Gln Met Asn Ser Glu Leu Ser Val Leu Ala 115 120 125 Asn Phe Ser GlnPro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu Asn 130 135 140 Val Tyr IleAsn Leu Thr Cys Ser Ser Ile His Gly Tyr Pro Glu Pro 145 150 155 160 LysLys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu Tyr 165 170 175Asp Gly Ile Met Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr Asp 180 185190 Val Ser Ile Ser Leu Ser Val Ser Phe Pro Asp Val Thr Ser Asn Met 195200 205 Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys Thr Arg Leu Leu Ser Ser210 215 220 Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro Asp HisGlu 225 230 235 240 Phe Gly Gly Ser Gly Gly Ser Ala Thr Pro Gln Asn IleThr Asp Leu 245 250 255 Cys Ala Glu Tyr His Asn Thr Gln Ile His Thr LeuAsn Asp Lys Ile 260 265 270 Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys ArgGlu Met Ala Ile Ile 275 280 285 Thr Phe Lys Asn Gly Ala Thr Phe Gln ValGlu Val Pro Gly Ser Gln 290 295 300 His Ile Asp Ser Gln Lys Lys Ala IleGlu Arg Met Lys Asp Thr Leu 305 310 315 320 Arg Ile Ala Tyr Leu Thr GluAla Lys Val Glu Lys Leu Cys Val Trp 325 330 335 Asn Asn Lys Thr Pro HisAla Ile Ala Ala Ile Ser Met Ala Asn 340 345 350 19 31 PRT ArtificialSequence Peptides 19 Ser Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu LysGlu Asn Ala 1 5 10 15 Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys GluLeu Ala Gln 20 25 30 20 30 PRT Artificial Sequence peptides 20 Ala GlnLeu Lys Lys Lys Leu Gln Ala Leu Lys Lys Lys Asn Ala Gln 1 5 10 15 LeuLys Gln Lys Leu Gln Ala Leu Lys Lys Lys Leu Ala Gln 20 25 30 21 1095 DNAArtificial Sequence Fusion constructs with human and bacterial sequences21 atggccataa gtggagtccc tgtgctagga tttttcatca tagctgtgct gatgagcgct 60caggaatcat gggctatcaa agaagaacat gtgatcatcc aggccgagtt ctatctgaat 120cctgaccaat caggcgagtt tatgtttgac tttgatggtg atgagatttt ccatgtggat 180atggcaaaga aggagacggt ctggcggctt gaagaatttg gacgatttgc cagctttgag 240gctcaaggtg cattggccaa catagctgtg gacaaagcca acctggaaat catgacaaag 300cgctccaact atactccgat caccaatgta cctccagagg taactgtgct cacgaacagc 360cctgtggaac tgagagagcc caacgtcctc atctgtttca tcgacaagtt caccccacca 420gtggtcaatg tcacgtggct tcgaaatgga aaacctgtca ccacaggagt gtcagagaca 480gtcttcctgc ccagggaaga ccaccttttc cgcaagttcc actatctccc cttcctgccc 540tcaactgagg acgtttacga ctgcagggtg gagcactggg gcttggatga gcctcttctc 600aagcactggg agfttgatgc tccaagccct ctcccagaga ctacagagga attcggtggt 660tccggtggtt ccgcgcagct ggaatgggaa ctgcaggcgc tggaaaaaga aaacgcgcag 720ctggaatggg aactgcaggc gctggaaaaa gaactggcgc agggcggctc cggtggtagc 780gccacacctc aaaatattac tgatttgtgt gcagaatacc acaacacaca aatacatacg 840ctaaatgata agatattttc gtatacagaa tctctagctg gaaaaagaga gatggctatc 900attactttta agaatggtgc aacttttcaa gtagaagtac caggtagtca acatatagat 960tcacaaaaaa aagcgattga aaggatgaag gataccctga ggattgcata tcttactgaa 1020gctaaagtcg aaaagttatg tgtatggaat aataaaacgc ctcatgcgat tgccgcaatt 1080agtatggcaa attaa 1095 22 364 PRT Artificial Sequence Fusion constructswith human and bacterial sequences 22 Met Ala Ile Ser Gly Val Pro ValLeu Gly Phe Phe Ile Ile Ala Val 1 5 10 15 Leu Met Ser Ala Gln Glu SerTrp Ala Ile Lys Glu Glu His Val Ile 20 25 30 Ile Gln Ala Glu Phe Tyr LeuAsn Pro Asp Gln Ser Gly Glu Phe Met 35 40 45 Phe Asp Phe Asp Gly Asp GluIle Phe His Val Asp Met Ala Lys Lys 50 55 60 Glu Thr Val Trp Arg Leu GluGlu Phe Gly Arg Phe Ala Ser Phe Glu 65 70 75 80 Ala Gln Gly Ala Leu AlaAsn Ile Ala Val Asp Lys Ala Asn Leu Glu 85 90 95 Ile Met Thr Lys Arg SerAsn Tyr Thr Pro Ile Thr Asn Val Pro Pro 100 105 110 Glu Val Thr Val LeuThr Asn Ser Pro Val Glu Leu Arg Glu Pro Asn 115 120 125 Val Leu Ile CysPhe Ile Asp Lys Phe Thr Pro Pro Val Val Asn Val 130 135 140 Thr Trp LeuArg Asn Gly Lys Pro Val Thr Thr Gly Val Ser Glu Thr 145 150 155 160 ValPhe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe His Tyr Leu 165 170 175Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg Val Glu His 180 185190 Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe Asp Ala Pro 195200 205 Ser Pro Leu Pro Glu Thr Thr Glu Glu Phe Gly Gly Ser Gly Gly Ser210 215 220 Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Asn AlaGln 225 230 235 240 Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu AlaGln Gly Gly 245 250 255 Ser Gly Gly Ser Ala Thr Pro Gln Asn Ile Thr AspLeu Cys Ala Glu 260 265 270 Tyr His Asn Thr Gln Ile His Thr Leu Asn AspLys Ile Phe Ser Tyr 275 280 285 Thr Glu Ser Leu Ala Gly Lys Arg Glu MetAla Ile Ile Thr Phe Lys 290 295 300 Asn Gly Ala Thr Phe Gln Val Glu ValPro Gly Ser Gln His Ile Asp 305 310 315 320 Ser Gln Lys Lys Ala Ile GluArg Met Lys Asp Thr Leu Arg Ile Ala 325 330 335 Tyr Leu Thr Glu Ala LysVal Glu Lys Leu Cys Val Trp Asn Asn Lys 340 345 350 Thr Pro His Ala IleAla Ala Ile Ser Met Ala Asn 355 360 23 861 DNA Artificial SequenceFusion constructs with human and bacterial sequences 23 atggtgtgtctgaagttccc tggaggctcc tgcatggcag ctctgacagt gacactgatg 60 gtgctgagctccccactggc tttggctggg gacacccgac cacgtttctt ggagcaggtt 120 aaacatgagtgtcatttctt caacgggacg gagcgggtgc ggttcctgga cagatacttc 180 tatcaccaagaggagtacgt gcgcttcgac agcgacgtgg gggagtaccg ggcggtgacg 240 gagctggggcggcctgatgc cgagtactgg aacagccaga aggacctcct ggagcagaag 300 cgggccgcggtggacaccta ctgcagacac aactacgggg ttggtgagag cttcacagtg 360 cagcggcgagtctatcctga ggtgactgtg tatcctgcaa agacccagcc cctgcagcac 420 cacaacctcctggtctgctc tgtgaatggt ttctatccag gcagcattga agtcaggtgg 480 ttccggaacggccaggaaga gaagactggg gtggtgtcca caggcctgat ccagaatgga 540 gactggaccttccagaccct ggtgatgctg gaaacagttc ctcggagtgg agaggtttac 600 acctgccaagtggagcaccc aagcctgacg agccctctca cagtggaatg gagagcacgg 660 tctgaatctgcacagagcaa gggcggctcc ggtggtagcg cccagctgaa gaagaaactc 720 caggctctgaaaaaaaagaa tgcccagctc aagcagaagc tgcaggccct gaagaaaaag 780 ctggctcagggttccggtgg ttccgcgggt ggtggtttga acgacatctt cgaagctc.ag 840 aaaatcgaatggcactaata a 861 24 285 PRT Artificial Sequence Fusion constructs withhuman and bacterial sequences 24 Met Val Cys Leu Lys Phe Pro Gly Gly SerCys Met Ala Ala Leu Thr 1 5 10 15 Val Thr Leu Met Val Leu Ser Ser ProLeu Ala Leu Ala Gly Asp Thr 20 25 30 Arg Pro Arg Phe Leu Glu Gln Val LysHis Glu Cys His Phe Phe Asn 35 40 45 Gly Thr Glu Arg Val Arg Phe Leu AspArg Tyr Phe Tyr His Gln Glu 50 55 60 Glu Tyr Val Arg Phe Asp Ser Asp ValGly Glu Tyr Arg Ala Val Thr 65 70 75 80 Glu Leu Gly Arg Pro Asp Ala GluTyr Trp Asn Ser Gln Lys Asp Leu 85 90 95 Leu Glu Gln Lys Arg Ala Ala ValAsp Thr Tyr Cys Arg His Asn Tyr 100 105 110 Gly Val Gly Glu Ser Phe ThrVal Gln Arg Arg Val Tyr Pro Glu Val 115 120 125 Thr Val Tyr Pro Ala LysThr Gln Pro Leu Gln His His Asn Leu Leu 130 135 140 Val Cys Ser Val AsnGly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp 145 150 155 160 Phe Arg AsnGly Gln Glu Glu Lys Thr Gly Val Val Ser Thr Gly Leu 165 170 175 Ile GlnAsn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr 180 185 190 ValPro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser 195 200 205Leu Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala 210 215220 Gln Ser Lys Gly Gly Ser Gly Gly Ser Ala Gln Leu Lys Lys Lys Leu 225230 235 240 Gln Ala Leu Lys Lys Lys

Asn Ala Gln Leu Lys Gln Lys Leu Gln Ala 245 250 255 Leu Lys Lys Lys LeuAla Gln Gly Ser Gly Gly Ser Ala Gly Gly Gly 260 265 270 Leu Asn Asp IlePhe Glu Ala Gln Lys Ile Glu Trp His 275 280 285

All patents and patent applications, publications, scientific articles,and other referenced materials mentioned in this specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each of which is hereby incorporated byreference to the same extent as if each reference had been incorporatedby reference in its entirety individually. Applicants reserve the rightto physically incorporate into this specification any and all materialsand information from any such patents and patent applications,publications, scientific articles, electronically available information,and other referenced materials or documents.

The specific methods and products described in this specification arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention, and it isunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims. Other objects, aspects, and embodiments will occur tothose skilled in the art upon consideration of this specification andare encompassed within the spirit of the invention as defined by thescope of the claims. It is understood that this invention is not limitedto the particular materials and methods described, and it will bereadily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Also, the terms“comprising”, “including”, “containing”, etc. are to be read expansivelyand without limitation. It must be noted that as used herein and in theappended claims, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude-any now-existing orlater-developed equivalent of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention as claimed. Thus, it will beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand/or variation of the disclosed elements may be resorted to by thoseskilled in the art, and that such modifications and variations arewithin the scope of the invention as claimed.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. An artificial antigen presenting cell, comprising: a) a liposomecomprising a fluid lipid bilayer; b) a plurality of MHC moleculesassociated with the lipid bilayer, wherein each MHC molecule is: i.selected from the group consiting of a MHC class I molecule, a MHC classII molecule, and a mixture of MHC class I molecules and MHC class IImolecules; and ii. comnplexed with an antigenic peptide; and c) aplurality of accessory molecules associated with the lipid bilayer. 2.An artificial antigen presenting cell according to claim 1 furthercomprising a plurality of co-stimulatory molecules associated with thelipid bilayer.
 3. An artificial antigen presenting cell according toclaim 1 further comprising a plurality of adhesion molecules associatedwith the lipid bilayer.
 4. An artificial antigen presenting cellaccording to claim 1 wherein the MHC molecules comprise an extracellularregion that binds the antigenic peptide and a transmembrane domain thatassociates with the lipid bilayer of the liposome.
 5. An artificialantigen presenting cell according to claim 1 wherein the MHC moleculesare associated with the lipid bilayer of the liposome by way of ananchor moiety embedded in the lipid bilayer.
 6. An artificial antigenpresenting cell, comprising: a) a scaffold; b) a plurality of MHCmolecules linked to the scaffold, wherein each MHC molecule is: i.selected from the group consiting of a MHC class I molecule and a MRCclass II molecule; and ii. complexed with an antigenic peptide; and c) aplurality of accessory molecules linked to the scaffold and disposed tostablize interactions between T cell receptors specific for the MHCmolecules complexed with the antigenic peptide.
 7. An artificial antigenpresenting cell according to claim 6 wherein the scaffold is selectedfrom the group consisting of a dendrimer and a particle.
 8. Anartificial antigen presenting cell according to claim 6 wherein the MHCmolecules are covalently linked to the scaffold.
 9. An artificialantigen presenting cell according to claim 8 wherein the MHC moleculesare linked to the scaffold through linkers.
 10. An artificial antigenpresenting cell according to claim 6 wherein the accessory molecules arecovalently linked to the scaffold.
 11. An artificial antigen presentingcell according to claim 10 wherein the accessory molecules are linked tothe scaffold through linkers.
 12. An artificial antigen presenting cellaccording to claim 6 further comprising an additional molecule selectedfrom the group consisting of a co-stimulatory molecule, an adhesionmolecule, and irrelevant molecule.
 13. A method of isolating anantigen-specific T cell, comprising: a) forming a T cell/aAPC complex bycontacting a sample comprising T cells with an artificial antigenpresenting cell according to claim 1, wherein the antigenic peptidecomplexed with the MHC molecule is the antigen against which theantigen-specific T cell is reactive; and b) isolating theantigen-specific T cell from the T cell/aAPC complex.
 14. A method ofisolating an antigen-specific T cell, comprising a) forming a Tcell/aAPC complex by contacting a sample comprising T cells with anartificial antigen presenting cell according to claim 6, wherein theantigenic peptide complexed with the MHC molecule is the antigen againstwhich the antigen-specific T cell is reactive; and b) isolating theantigen-specific T cell from the T cell/aAPC complex.
 15. A method ofmodulating T cell activity, comprising forming a T cell/aAPC complex bycontacting T cells with an artificial antigen presenting cell accordingto claim 1, wherein the antigenic peptide loaded on the MHC molecule isthe antigen against which an antigen-specific T cell is reactive,thereby modulating the activity of the antigen-specific T cell.
 16. Amethod according to claim 15 that is performed ex vivo.
 17. A methodaccording to claim 15 that is performed in vivo.
 18. A method ofmodulating T cell activity, comprising forming a T cell/aAPC complex bycontacting T cells with an artificial antigen presenting cell accordingto claim 6, wherein the antigenic peptide loaded on the MHC molecule isthe antigen against which an antigen-specific T cell is reactive,thereby modulating the activity of the antigen-specific T cell.
 19. Amethod according to claim 18 that is performed ex vivo.
 20. A methodaccording to claim 18 that is performed in vivo.